Fibroblast growth factor receptor 1 inhibition suppresses pancreatic cancer chemoresistance and chemotherapy-driven aggressiveness.

AIMS: Pancreatic ductal adenocarcinoma (PDAC) is often intrinsically-resistant to standard-of-care chemotherapies such as gemcitabine. Acquired gemcitabine resistance (GemR) can arise from treatment of initially-sensitive tumors, and chemotherapy can increase tumor aggressiveness. We investigated the molecular mechanisms of chemoresistance and chemotherapy-driven tumor aggressiveness, which are understood incompletely. METHODS: Differential proteomic analysis was employed to investigate chemotherapy-driven chemoresistance drivers and responses of PDAC cells and patient-derived tumor xenografts (PDX) having different chemosensitivities. We also investigated the prognostic value of FGFR1 expression in the efficacy of selective pan-FGFR inhibitor (FGFRi)-gemcitabine combinations. RESULTS: Quantitative proteomic analysis of a highly-GemR cell line revealed fibroblast growth factor receptor 1 (FGFR1) as the highest-expressed receptor tyrosine kinase. FGFR1 knockdown or FGFRi co-treatment enhanced gemcitabine efficacy and decreased GemR marker expression, implicating FGFR1 in augmentation of GemR. FGFRi treatment reduced PDX tumor progression and prolonged survival significantly, even in highly-resistant tumors in which neither single-agent showed efficacy. Gemcitabine exacerbated aggressiveness of highly-GemR tumors, based upon proliferation and metastatic markers. Combining FGFRi with gemcitabine or gemcitabine+nab-paclitaxel reversed tumor aggressiveness and progression, and prolonged survival significantly. In multiple PDAC PDXs, FGFR1 expression correlated with intrinsic tumor gemcitabine sensitivity. CONCLUSION: FGFR1 drives chemoresistance and tumor aggressiveness, which FGFRi can reverse.

Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current-based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and 'drug response' mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.

Species-Deconvolved Proteomics for In Situ Investigation of Tumor-Stroma Interactions after Treatment of Pancreatic Cancer Patient-Derived Xenografts with Combined Gemcitabine and Paclitaxel.

Tumor-stroma interactions are critical in pancreatic ductal adenocarcinoma (PDAC) progression and therapeutics. Patient-derived xenograft (PDX) models recapitulate tumor-stroma interactions, but the conventional antibody-based immunoassay is inadequate to discriminate tumor and stromal proteins. Here, we describe a species-deconvolved proteomics approach embedded in IonStar that can unambiguously quantify the tumor (human-derived) and stromal (mouse-derived) proteins in PDX samples, enabling unbiased investigation of tumor and stromal proteomes with excellent quantitative reproducibility. With this strategy, we studied tumor-stroma interactions in PDAC PDXs that responded differently to Gemcitabine combined with nab-Paclitaxel (GEM+PTX) treatment. By analyzing 48 PDX animals 24 h/192 h after treatment with/without GEM+PTX, we quantified 7262 species-specific proteins under stringent cutoff criteria, with high reproducibility. For the PDX sensitive to GEM+PTX, the drug-dysregulated proteins in tumor cells were involved in suppressed oxidative phosphorylation and the TCA cycle, and in the stroma, inhibition of glycolytic activity was predominant, suggesting a relieved reverse Warburg effect by the treatment. In GEM+PTX-resistant PDXs, protein changes suggested extracellular matrix deposition and activation of tumor cell proliferation. Key findings were validated by immunohistochemistry (IHC). Overall, this approach provides a species-deconvolved proteomic platform that could advance cancer therapeutic studies by enabling unbiased exploration of tumor-stroma interactions in the large number of PDX samples required for such investigations.

Highly Reproducible Quantitative Proteomics Analysis of Pancreatic Cancer Cells Reveals Proteome-Level Effects of a Novel Combination Drug Therapy That Induces Cancer Cell Death via Metabolic Remodeling and Activation of the Extrinsic Apoptosis Pathway.

Pancreatic cancer patients have poor survival rates and are frequently treated using gemcitabine (Gem). However, initial tumor sensitivity often gives way to rapid development of resistance. Gem-based drug combinations are employed to increase efficacy and mitigate resistance, but our understanding of molecular-level drug interactions, which could assist in the development of more effective therapeutic regimens, is limited. Global quantitative proteomic analysis could provide novel mechanistic insights into drug combination interactions, but it is challenging to achieve high-quality quantitative proteomics analysis of the large sample sets that are typically required for drug combination studies. Here, we investigated molecular-level temporal interactions of Gem with BGJ398 (infigratinib), a recently approved pan-FGFR inhibitor, in multiple treatment groups (N = 42 samples) using IonStar, a robust large-scale proteomics method that employs well-controlled, ultrahigh-resolution MS1 quantification. A total of 5514 proteins in the sample set were quantified without missing data, requiring >2 unique peptides/protein, <1% protein false discovery rate (FDR), <0.1% peptide FDR, and CV < 10%. Functional analysis of the differentially altered proteins revealed drug-dysregulated processes such as metabolism, apoptosis, and antigen presentation pathways. These changes were validated experimentally using Seahorse metabolic assays and immunoassays. Overall, in-depth analysis of large-scale proteomics data provided novel insights into possible mechanisms by which FGFR inhibitors complement and enhance Gem activity in pancreatic cancers.

Synergistic Pharmacodynamic Effects of Gemcitabine and Fibroblast Growth Factor Receptor Inhibitors on Pancreatic Cancer Cell Cycle Kinetics and Proliferation.

Median survival of pancreatic ductal adenocarcinoma cancer (PDAC) is 6 months, with 9% 5-year survival. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and combination therapies integrating novel targeted agents could improve outcomes. Fibroblast growth factor (FGF) receptors (FGFRs) play important roles in PDAC growth and invasion. Therefore, FGFR inhibitors (FGFRi) merit further investigation. Efficacy of Gem combined with NVP-BGJ398, a pan-FGFRi, was investigated in multiple PDAC cell lines exposed to the drugs alone and combined. Cell cycle distribution and cell numbers were quantified over time. Two pharmacodynamic models were developed to investigate Gem/BGJ398 interactions quantitatively: a drug-mediated cell proliferation/death model, and a drug-perturbed cell cycle progression model. The models captured temporal changes in cell numbers, cell cycle progression, and cell death during drug exposure. Simultaneous fitting of all data provided reasonable parameter estimates. Therapeutic efficacy was then evaluated in a PDAC mouse model. Compared with Gem alone, combined Gem + FGFRi significantly downregulated ribonucleotide-diphosphate reductase large subunit 1 (RRM1), a gemcitabine resistance (GemR) biomarker, suggesting the FGFRi inhibited GemR emergence. The cell proliferation/death pharmacodynamic model estimated the drug interaction coefficient ψ death = 0.798, suggesting synergistic effects. The mechanism-based cell cycle progression model estimated drug interaction coefficient ψ cycle = 0.647, also suggesting synergy. Thus, FGFR inhibition appears to synergize with Gem in PDAC cells and tumors by sensitizing cells to Gem-mediated inhibition of proliferation and cell cycle progression. SIGNIFICANCE STATEMENT: An integrated approach of quantitative modeling and experimentation was employed to investigate the nature of fibroblast growth factor receptor inhibitor (FGFRi)/gemcitabine (Gem) interaction, and to identify mechanisms by which FGFRi exposure reverses Gem resistance in pancreatic cancer cells. The results show that FGFRi interacts synergistically with Gem to sensitize pancreatic cancer cells and tumors to Gem-mediated inhibition of proliferation and cell cycle progression. Thus, addition of FGFRi to standard-of-care Gem treatment could be a clinically deployable approach to enhance therapeutic benefit to pancreatic cancer patients.

Controlled coupling of an ultrapotent auristatin warhead to cetuximab yields a next-generation antibody-drug conjugate for EGFR-targeted therapy of KRAS mutant pancreatic cancer.

BACKGROUND: Antibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC. METHODS: In vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens. RESULTS: Site-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells. CONCLUSIONS: Together, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

Pharmacodynamic modeling of synergistic birinapant/paclitaxel interactions in pancreatic cancer cells.

BACKGROUND: For most patients, pancreatic adenocarcinoma responds poorly to treatment, and novel therapeutic approaches are needed. Standard-of-care paclitaxel (PTX), combined with birinapant (BRP), a bivalent mimetic of the apoptosis antagonist SMAC (second mitochondria-derived activator of caspases), exerts synergistic killing of PANC-1 human pancreatic adenocarcinoma cells. METHODS: To investigate potential mechanisms underlying this synergistic pharmacodynamic interaction, data capturing PANC-1 cell growth, apoptosis kinetics, and cell cycle distribution were integrated with high-quality IonStar-generated proteomic data capturing changes in the relative abundance of more than 3300 proteins as the cells responded to the two drugs, alone and combined. RESULTS: PTX alone (15 nM) elicited dose-dependent G2/M-phase arrest and cellular polyploidy. Combined BRP/PTX (150/15 nM) reduced G2/M by 35% and polyploid cells by 45%, and increased apoptosis by 20%. Whereas BRP or PTX alone produced no change in the pro-apoptotic protein pJNK, and a slight increase in the anti-apoptotic protein Bcl2, the drug combination increased pJNK and decreased Bcl2 significantly compared to the vehicle control. A multi-scale, mechanism-based mathematical model was developed to investigate integrated birinapant/paclitaxel effects on temporal profiles of key proteins involved in kinetics of cell growth, death, and cell cycle distribution. CONCLUSIONS: The model, consistent with the observed reduction in the Bcl2/BAX ratio, suggests that BRP-induced apoptosis of mitotically-arrested cells is a major contributor to the synergy between BRP and PTX. Coupling proteomic and cellular response profiles with multi-scale pharmacodynamic modeling provides a quantitative mechanistic framework for evaluating pharmacodynamically-based drug-drug interactions in combination chemotherapy, and could potentially guide the development of promising drug regimens.

Temporal Effects of Combined Birinapant and Paclitaxel on Pancreatic Cancer Cells Investigated via Large-Scale, Ion-Current-Based Quantitative Proteomics (IonStar).

Despite decades of effort, pancreatic adenocarcinoma (PDAC) remains an intractable clinical challenge. An insufficient understanding of mechanisms underlying tumor cell responses to chemotherapy contributes significantly to the lack of effective treatment regimens. Here, paclitaxel, a first-line chemotherapeutic agent, was observed to interact synergistically with birinapant, a second mitochondrial-derived activator of caspases mimetic. Therefore, we investigated molecular-level drug interaction mechanisms using comprehensive, reproducible, and well-controlled ion-current-based MS1 quantification (IonStar). By analyzing 40 biological samples in a single batch, we compared temporal proteomic responses of PDAC cells treated with birinapant and paclitaxel, alone and combined. Using stringent criteria (e.g. strict false-discovery-rate (FDR) control, two peptides/protein), we quantified 4069 unique proteins confidently (99.8% without any missing data), and 541 proteins were significantly altered in the three treatment groups, with an FDR of <1%. Interestingly, most of these proteins were altered only by combined birinapant/paclitaxel, and these predominantly represented three biological processes: mitochondrial function, cell growth and apoptosis, and cell cycle arrest. Proteins responsible for activation of oxidative phosphorylation, fatty acid ß-oxidation, and inactivation of aerobic glycolysis were altered largely by combined birinapant/paclitaxel compared with single drugs, suggesting the Warburg effect, which is critical for survival and proliferation of cancer cells, was alleviated by the combination treatment. Metabolic profiling was performed to confirm substantially greater suppression of the Warburg effect by the combined agents compared with either drug alone. Immunoassays confirmed proteomic data revealing changes in apoptosis/survival signaling pathways, such as inhibition of PI3K/AKT, JAK/STAT, and MAPK/ERK signal transduction, as well as induction of G2/M arrest, and showed the drug combination induced much more apoptosis than did single agents. Overall, this in-depth, large-scale proteomics study provided novel insights into molecular mechanisms underlying synergy of combined birinapant/paclitaxel and describes a proteomics/informatics pipeline that can be applied broadly to the development of cancer drug combination regimens.

Establishment and Characterisation by Expression Microarray of Patient-Derived Xenograft Panel of Human Pancreatic Adenocarcinoma Patients.

Pancreatic cancer remains among the most lethal cancers worldwide, with poor early detection rates and poor survival rates. Patient-derived xenograft (PDX) models have increasingly been used in preclinical and clinical research of solid cancers to fulfil unmet need. Fresh tumour samples from human pancreatic adenocarcinoma patients were implanted in severe combined immunodeficiency (SCID) mice. Samples from 78% of treatment-naïve pancreatic ductal adenocarcinoma patients grew as PDX tumours and were confirmed by histopathology. Frozen samples from F1 PDX tumours could be later successfully passaged in SCID mice to F2 PDX tumours. The human origin of the PDX was confirmed using human-specific antibodies; however, the stromal component was replaced by murine cells. Cell lines were successfully developed from three PDX tumours. RNA was extracted from eight PDX tumours and where possible, corresponding primary tumour (T) and adjacent normal tissues (N). mRNA profiles of tumour vs. F1 PDX and normal vs. tumour were compared by Affymetrix microarray analysis. Differential gene expression showed over 5000 genes changed across the N vs. T and T vs. PDX samples. Gene ontology analysis of a subset of genes demonstrated genes upregulated in normal vs. tumour vs. PDX were linked with cell cycle, cycles cell process and mitotic cell cycle. Amongst the mRNA candidates elevated in the PDX and tumour vs. normal were SERPINB5, FERMT1, AGR2, SLC6A14 and TOP2A. These genes have been associated with growth, proliferation, invasion and metastasis in pancreatic cancer previously. Cumulatively, this demonstrates the applicability of PDX models and transcriptomic array to identify genes associated with growth and proliferation of pancreatic cancer.

Development and Evaluation of Tri-Functional Immunoliposomes for the Treatment of HER2 Positive Breast Cancer.

PURPOSE: Trastuzumab combined with Doxorubicin (DOX) demonstrates significant clinical activity in human epidermal growth factor receptor-2 (HER2)-positive breast cancer (BC). However, emergence of treatment resistance and trastuzumab associated cardiotoxicity remain clinical challenges. In an effort to improve patient outcome, we have developed and evaluated novel tri-functional immunoliposomes (TFIL) that target HER2-receptors on BC cells and CD3-receptors on T-lymphocytes, and deliver DOX. METHODS: Trastuzumab (anti-HER2) and OKT-3 (anti-CD3) antibodies were conjugated to liposomes using a micelle-transfer method. Cytotoxicity of targeted immunoliposomes loaded with DOX was examined in vitro on HER2-positive BC cells (BT474), with peripheral blood monocytic cells (PBMC) as immune effector cells. RESULTS: TFIL demonstrated high antibody-liposome conjugation ratios (100-130 µg protein/µmol phospholipid) and cargo capacity (0.21 mol:mol drug:lipid), highly efficient DOX loading (>90%), a particle size favorable for extended circulation (~150 nm), and good stability (up to 3 months at 4°C). In the presence of PBMCs, TFIL showed complete killing of BT474 cells, and were superior to mono-targeted trastuzumab-bearing liposomes, non-targeted liposomes, and free Trastuzumab and DOX. CONCLUSIONS: Novel anti-HER2xCD3 + DOX TFIL show promise as a means to both engage immune cells against HER2 positive breast cancer cells and deliver chemotherapy, and have the potential to improve clinical outcomes.

Physiologically-based pharmacokinetic and pharmacodynamic models for gemcitabine and birinapant in pancreatic cancer xenografts.

The anticancer effects of combined gemcitabine and birinapant were demonstrated as synergistic in PANC-1 cells in vitro. In this study, pharmacokinetic information derived from experiments and the literature was utilized to develop full physiologically-based pharmacokinetic (PBPK) models that characterize individual drugs. The predicted intra-tumor drug concentrations were used as the driving force within a linked PBPK/PD model for treatment-mediated changes in tumor volume in a xenograft mouse model. The efficacy of the drug combination in vivo was evaluated mathematically as exhibiting additivity. The network model developed for drug effects in the in vitro cell cultures was applied successfully to link the in vivo tumor drug concentrations with tumor growth inhibition, incorporating more mechanistic features and accounting for disparate drug interaction outcomes in vitro and in vivo.

Flow arrest intra-arterial delivery of small TAT-decorated and neutral micelles to gliomas.

The cell-penetrating trans-activator of transcription (TAT) is a cationic peptide derived from human immunodeficiency virus-1. It has been used to facilitate macromolecule delivery to various cell types. This cationic peptide is capable of crossing the blood-brain barrier and therefore might be useful for enhancing the delivery of drugs that target brain tumors. Here we test the efficiency with which relatively small (20 nm) micelles can be delivered by an intra-arterial route specifically to gliomas. Utilizing the well-established method of flow-arrest intra-arterial injection we compared the degree of brain tumor deposition of cationic TAT-decorated micelles versus neutral micelles. Our in vivo and post-mortem analyses confirm glioma-specific deposition of both TAT-decorated and neutral micelles. Increased tumor deposition conferred by the positive charge on the TAT-decorated micelles was modest. Computational modeling suggested a decreased relevance of particle charge at the small sizes tested but not for larger particles. We conclude that continued optimization of micelles may represent a viable strategy for targeting brain tumors after intra-arterial injection. Particle size and charge are important to consider during the directed development of nanoparticles for intra-arterial delivery to brain tumors.

Large-Scale, Ion-Current-Based Proteomic Investigation of the Rat Striatal Proteome in a Model of Short- and Long-Term Cocaine Withdrawal.

Given the tremendous detriments of cocaine dependence, effective diagnosis and patient stratification are critical for successful intervention yet difficult to achieve due to the largely unknown molecular mechanisms involved. To obtain new insights into cocaine dependence and withdrawal, we employed a reproducible, reliable, and large-scale proteomics approach to investigate the striatal proteomes of rats (n = 40, 10 per group) subjected to chronic cocaine exposure, followed by either short- (WD1) or long- (WD22) term withdrawal. By implementing a surfactant-aided precipitation/on-pellet digestion procedure, a reproducible and sensitive nanoLC-Orbitrap MS analysis, and an optimized ion-current-based MS1 quantification pipeline, >2000 nonredundant proteins were quantified confidently without missing data in any replicate. Although cocaine was cleared from the body, 129/37 altered proteins were observed in WD1/WD22 that are implicated in several biological processes related closely to drug-induced neuroplasticity. Although many of these changes recapitulate the findings from independent studies reported over the last two decades, some novel insights were obtained and further validated by immunoassays. For example, significantly elevated striatal protein kinase C activity persisted over the 22 day cocaine withdrawal. Cofilin-1 activity was up-regulated in WD1 and down-regulated in WD22. These discoveries suggest potentially distinct structural plasticity after short- and long-term cocaine withdrawal. In addition, this study provides compelling evidence that blood vessel narrowing, a long-known effect of cocaine use, occurred after long-term but not short-term withdrawal. In summary, this work developed a well-optimized paradigm for ion-current-based quantitative proteomics in brain tissues and obtained novel insights into molecular alterations in the striatum following cocaine exposure and withdrawal.

Cationizable lipid micelles as vehicles for intraarterial glioma treatment.

The relative abundance of anionic lipids on the surface of endothelia and on glioma cells suggests a workable strategy for selective drug delivery by utilizing cationic nanoparticles. Furthermore, the extracellular pH of gliomas is relatively acidic suggesting that tumor selectivity could be further enhanced if nanoparticles can be designed to cationize in such an environment. With these motivating hypotheses the objective of this study was to determine whether nanoparticulate (20 nm) micelles could be designed to improve their deposition within gliomas in an animal model. To test this, we performed intra-arterial injection of micelles labeled with an optically quantifiable dye. We observed significantly greater deposition (end-tissue concentration) of cationizable micelles as compared to non-ionizable micelles in the ipsilateral hemisphere of normal brains. More importantly, we noted enhanced deposition of cationizable as compared to non-ionizable micelles in glioma tissue as judged by semiquantitative fluorescence analysis. Micelles were generally able to penetrate to the core of the gliomas tested. Thus we conclude that cationizable micelles may be constructed as vehicles for facilitating glioma-selective delivery of compounds after intraarterial injection.

Mechanism-based mathematical modeling of combined gemcitabine and birinapant in pancreatic cancer cells.

Combination chemotherapy is standard treatment for pancreatic cancer. However, current drugs lack efficacy for most patients, and selection and evaluation of new combination regimens is empirical and time-consuming. The efficacy of gemcitabine, a standard-of-care agent, combined with birinapant, a pro-apoptotic antagonist of Inhibitor of Apoptosis Proteins (IAPs), was investigated in pancreatic cancer cells. PANC-1 cells were treated with vehicle, gemcitabine (6, 10, 20 nM), birinapant (50, 200, 500 nM), and combinations of the two drugs. Temporal changes in cell numbers, cell cycle distribution, and apoptosis were measured. A basic pharmacodynamic (PD) model based on cell numbers, and a mechanism-based PD model integrating all measurements, were developed. The basic PD model indicated that synergistic effects occurred in both cell proliferation and death processes. The mechanism-based model captured key features of drug action: temporary cell cycle arrest in S phase induced by gemcitabine alone, apoptosis induced by birinapant alone, and prolonged cell cycle arrest and enhanced apoptosis induced by the combination. A drug interaction term Ψ was employed in the models to signify interactions of the combination when data were limited. When more experimental information was utilized, Ψ values approaching 1 indicated that specific mechanisms of interactions were captured better. PD modeling identified the potential benefit of combining gemcitabine and birinapant, and characterized the key interaction pathways. An optimal treatment schedule of pretreatment with gemcitabine for 24-48 h was suggested based on model predictions and was verified experimentally. This approach provides a generalizable modeling platform for exploring combinations of cytostatic and cytotoxic agents in cancer cell culture studies.

Transient cerebral hypoperfusion assisted intraarterial cationic liposome delivery to brain tissue.

Transient cerebral hypoperfusion (TCH) has empirically been used to assist intraarterial (IA) drug delivery to brain tumors. Transient (<3 min) reduction of cerebral blood flow (CBF) occurs during many neuro- and cardiovascular interventions and has recently been used to better target IA drugs to brain tumors. In the present experiments, we assessed whether the effectiveness of IA delivery of cationic liposomes could be improved by TCH. Cationic liposomes composed of 1:1 DOTAP:PC (dioleoyl-trimethylammonium-propane:phosphatidylcholine) were administered to three groups of Sprague-Dawley rats. In the first group, we tested the effect of blood flow reduction on IA delivery of cationic liposomes. In the second group, we compared TCH-assisted IA liposomal delivery versus intravenous (IV) administration of the same dose. In the third group, we assessed retention of cationic liposomes in brain 4 h after TCH assisted delivery. The liposomes contained a near infrared dye, DilC18(7), whose concentration could be measured in vivo by diffuse reflectance spectroscopy. IA injections of cationic liposomes during TCH increased their delivery approximately fourfold compared to injections during normal blood flow. Optical pharmacokinetic measurements revealed that relative to IV injections, IA injection of cationic liposomes during TCH produced tissue concentrations that were 100-fold greater. The cationic liposomes were retained in the brain tissue 4 h after a single IA injection. There was no gross impairment of neurological functions in surviving animals. Transient reduction in CBF significantly increased IA delivery of cationic liposomes in the brain. High concentrations of liposomes could be delivered to brain tissue after IA injections with concurrent TCH while none could be detected after IV injection. IA-TCH injections were well tolerated and cationic liposomes were retained for at least 4 h after IA administration. These results should encourage development of cationic liposomal formulations of chemotherapeutic drugs and their IA delivery during TCH.

Cationic surface charge enhances early regional deposition of liposomes after intracarotid injection.

Rapid first pass uptake of drugs is necessary to increase tissue deposition after intraarterial (IA) injection. Here we tested whether brain tissue deposition of a nanoparticulate liposomal carrier could be enhanced by coordinated manipulation of liposome surface charge and physiological parameters, such as IA injection during transient cerebral hypoperfusion (TCH). Different degrees of blood-brain barrier disruption were induced by focused ultrasound in three sets of Sprague-Dawley rats. Brain tissue retention was then compared for anionic, cationic, and charge-neutral liposomes after IA injection combined with TCH. The liposomes contained a non-exchangeable carbocyanine membrane optical label that could be quantified using diffuse reflectance spectroscopy (DRS) or visualized by multispectral imaging. Real-time concentration-time curves in brain were obtained after each liposomal injection. Having observed greater tissue retention of cationic liposomes compared to other liposomes in all three groups, we tested uptake of cationic liposomes in C6 tumor bearing rats. DRS and multispectral imaging of postmortem sections revealed increased liposomal uptake by the C6 brain tumor as compared to non-tumor contralateral hemisphere. We conclude that regional deposition of liposomes can be enhanced without BBB disruption using IA injection of cationic liposomal formulations in healthy and C6 tumor bearing rats.

Integrated PK/PD Modeling Relates Smoothened Inhibitor Biomarkers to The Heterogeneous Intratumor Disposition of Cetuximab in Pancreatic Cancer Tumor Models.

Therapeutic antibodies have shown little efficacy in the treatment of pancreatic ductal adenocarcinomas (PDAC). Tumor desmoplasia, hypovascularity, and poor perfusion result in insufficient tumor cell exposure, contributing to treatment failure. Smoothened inhibitors of hedgehog signaling (sHHi) increase PDAC tumor permeability, perfusion, and drug delivery, and provide a tool to develop a quantitative, mechanistic understanding as to how the temporal dynamics of tumor priming can impact intratumor distribution of monoclonal antibodies (mAb). A linked pharmacokinetic (PK)/pharmacodynamic (PD) model was developed to integrate the plasma and tumor PK of a sHHi priming agent with its effects upon downstream stromal biomarkers Gli1, hyaluronic acid, and interstitial fluid pressure in PDAC patient-derived xenograft (PDX) tumors. In parallel, in situ tumor concentrations of cetuximab (CTX: anti-epidermal growth factor receptor; EGFR) were quantified as a marker for tumor delivery of mAb or antibody-drug conjugates. A minimal, physiologically-based pharmacokinetic (mPBPK) model was constructed to link sHHi effects upon mechanistic effectors of tumor barrier compromise with the intratumor distribution of CTX, and CTX occupancy of EGFR in tumors. Integration of the mPBPK model of mAb deposition and intratumor distribution with the PK/PD model of tumor responses to priming not only identified physiological parameters that are critical for tumor antibody distribution, but also provides insight into dosing regimens that could achieve maximal tumor disposition of therapeutic antibodies under conditions of transient PDAC tumor permeability barrier compromise that mechanistically-diverse tumor priming strategies may achieve.

Expression of fibroblast growth factor receptor 1 correlates inversely with the efficacy of single-agent fibroblast growth factor receptor-specific inhibitors in pancreatic cancer.

BACKGROUND AND PURPOSE: Elevated fibroblast growth factor receptor (FGFR) activity correlates with pancreatic adenocarcinoma (PDAC) progression and poor prognosis. However, its potential as a therapeutic target remains largely unexplored. EXPERIMENTAL APPROACH: The mechanisms of action and therapeutic effects of selective pan-FGFR inhibitors (pan-FGFRi) were explored using in vitro and in vivo PDAC models ranging from gemcitabine-sensitive to highly gemcitabine-resistant (GemR). Gain-/loss-of-function investigations were employed to define the role of individual FGFRs in cell proliferation, migration, and treatment response and resistance. RESULTS: The pan-FGFRi NVP-BGJ398 significantly inhibited cell proliferation, migration, and invasion, and downregulated key cell survival- and invasiveness markers in multiple PDAC cell lines. Gemcitabine is a standard-of-care for PDAC, but development of resistance to gemcitabine (GemR) compromises its efficacy. Acquired GemR was modelled experimentally by developing highly GemR cells using escalating gemcitabine exposure in vitro and in vivo. FGFRi treatment inhibited GemR cell proliferation, migration, GemR marker expression, and tumour progression. FGFR2 or FGFR3 loss-of-function by shRNA knockdown failed to decrease cell growth, whereas FGFR1 knockdown was lethal. FGFR1 overexpression promoted cell migration more than proliferation, and reduced FGFRi-mediated inhibition of proliferation and migration. Single-agent FGFRi suppressed the viability and growth of multiple patient-derived xenografts inversely with respect to FGFR1 expression, underscoring the influence of FGFR1-dependent tumour responses to FGFRi. Importantly, secondary data analysis showed that PDAC tumours expressed FGFR1 at lower levels than in normal pancreas tissue. CONCLUSIONS AND IMPLICATIONS: Single-agent FGFR inhibitors mediate selective, molecularly-targeted suppression of PDAC proliferation, and their effects are greatest in PDAC tumours expressing low-to-moderate levels of FGFR1.

Systems Pharmacodynamic Model of Combined Gemcitabine and Trabectedin in Pancreatic Cancer Cells. Part II: Cell Cycle, DNA Damage Response, and Apoptosis Pathways.

Despite decades of research efforts, pancreatic adenocarcinoma (PDAC) continues to present a formidable clinical challenge, demanding innovative therapeutic approaches. In a prior study, we reported the synergistic cytotoxic effects of gemcitabine and trabectedin on pancreatic cancer cells. To investigate potential mechanisms underlying this synergistic pharmacodynamic interaction, liquid chromatography-mass spectrometry-based proteomic analysis was performed, and a systems pharmacodynamics model (SPD) was developed to capture pancreatic cancer cell responses to gemcitabine and trabectedin, alone and combined, at the proteome level. Companion report Part I describes the proteomic workflow and drug effects on the upstream portion of the SPD model related to cell growth and migration, specifically the RTK-, integrin-, GPCR-, and calcium-signaling pathways. This report presents Part II of the SPD model. Here we describe drug effects on pathways associated with cell cycle, DNA damage response (DDR), and apoptosis, and provide insights into underlying mechanisms. Drug combination effects on protein changes in the cell cycle- and apoptosis pathways contribute to the synergistic effects observed between gemcitabine and trabectedin. The SPD model was subsequently incorporated into our previously-established cell cycle model, forming a comprehensive, multi-scale quantification platform for evaluating drug effects across multiple scales, spanning the proteomic-, cellular-, and subcellular levels. This approach provides a quantitative mechanistic framework for evaluating drug-drug interactions in combination chemotherapy, and could potentially serve as a tool to predict combinatorial efficacy and assist in target selection.