Model Of Pancreatic Adenocarcinoma Research Articles

Combination of farnesyl-transferase inhibition with KRAS G12D targeting breaks down therapeutic resistance in pancreatic cancer

Pancreatic adenocarcinoma is one of the deadliest forms of cancer with no effective therapeutic options. A KRAS mutation can be found in up to 90% of all pancreatic tumors, making it a promising therapeutic target. The introduction of new KRAS inhibitors has been a milestone in the history of KRAS mutant tumors; however, therapeutic resistance limits their efficacy. Thus, new therapeutic options, including combination therapies, are urgently needed. Recently, we have shown that KRAS G12C inhibitors in combination with farnesyl-transferase inhibitors exert synergistic antitumor effects. Here, we provide evidence for the feasibility of this combinational approach to break down resistance in KRAS G12D mutant pancreatic cancer. Although we have shown that the 3D environment dramatically sensitizes cells to MRTX1133 treatment, the synergistic effect of this drug combination is present in both 2D and 3D in the PANC1 pancreatic adenocarcinoma model, which showed high resistance to MRTX1133 in 2D. The effects of the combination treatment show an association with the inhibition of farnesylated regulatory proteins, including HRAS and RHEB, along with the expression level of KRAS. Our study warrants further investigation for the potential applicability of KRAS G12D inhibitors in combination with farnesyl-transferase inhibitors for the treatment of KRAS mutant pancreatic adenocarcinoma.

Nanoparticles use magnetoelectricity to target and eradicate cancer cells.

We investigated the theranostic capabilities of magnetoelectric nanoparticles (MENPs) combined with MRI via a murine model of pancreatic adenocarcinoma. MENPs leverage the magnetoelectric effect to convert an applied magnetic field into local electric fields, which can induce irreversible electroporation of tumor cell membranes when activated by MRI. Additionally, MENPs modulate MRI relaxivity, which can be used to predict the degree of tumor ablation. Through a pilot study (n=21) and a confirmatory study (n=27), we demonstrated that, ≥300 µg of MRI-activated MENPs significantly reduced tumor volumes, averaging a three-fold decrease as compared to controls. Furthermore, there was a direct correlation between the reduction in tumor T 2 relaxation times and tumor volume reduction, highlighting the predictive prognostic value of MENPs. Six of 17 mice in the confirmatory study's experimental arms achieved a durable complete response, showcasing the potential for durable treatment outcomes. Importantly, the administration of MENPs was not associated with any evident toxicities. This study presents the first in vivo evidence of an externally controlled, MRI-based, theranostic agent that effectively targets and treats solid tumors via irreversible electroporation while sparing normal tissues, offering a new and promising approach to cancer therapy.

A mathematical model for pancreatic cancer during intraepithelial neoplasia.

Cancer is the result of complex interactions of intrinsic and extrinsic cell processes, which promote sustained proliferation, resistance to apoptosis, reprogramming and reorganization. The evolution of any type of cancer emerges from the role of the microenvironmental conditions and their impact of some molecular complexes on certain signalling pathways. The understanding of the early onset of cancer requires a multiscale analysis of the cellular microenvironment. In this paper, we analyse a qualitative multiscale model of pancreatic adenocarcinoma by modelling the cellular microenvironment through elastic cell interactions and their intercellular communication mechanisms, such as growth factors and cytokines. We focus on the low-grade dysplasia (PanIN 1) and moderate dysplasia (PanIN 2) stages of pancreatic adenocarcinoma. To this end, we propose a gene-regulatory network associated with the processes of proliferation and apoptosis of pancreatic cells and its kinetics in terms of delayed differential equations to mimic cell development. Likewise, we couple the cell cycle with the spatial distribution of cells and the transport of growth factors to show that the adenocarcinoma evolution is triggered by inflammatory processes. We show that the oncogene RAS may be an important target for developing anti-inflammatory strategies that limit the emergence of more aggressive adenocarcinomas.

Reversible chemoresistance of pancreatic cancer grown as spheroids

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

UPF1 deficiency enhances mitochondrial ROS which promotes an immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

Upstream frameshift 1 (UPF1) is an RNA helicase involved in a number of mRNA regulatory processes including nonsense-mediated decay. Mutations in the UPF1 locus that reduce its expression have been associated with adenosquamous carcinoma of the pancreas, a particularly aggressive form of the disease. To determine the effect of Upf1 suppression in a murine model of pancreatic adenocarcinoma, we silenced with shRNA Upf1 in cells derived from an autochthonous tumor in an LSL-Kras G12D/+ ; Trp53 R172H/+ ; Pdx-1 Cre/+ mouse (KPC) and orthotopically implanted these cells in the pancreas of C57BL/6 mice. Tumors derived from Upf1-deficient cells were markedly larger than those derived from control cells, a difference observed only in immunocompetent mice. The immune infiltrate of Upf1-deficient tumors was enriched in myeloid-derived suppressor cells (MDSCs) and depleted of CD8 + cells compared to control KPC tumors. Upf1-deficient KPC cells secreted inflammatory cytokines including G-CSF and CXCL2, known to recruit MDSCs. Cytokine secretion from Upf1-deficient KPC cells was induced by increased levels of mitochondrial reactive oxygen species (ROS), which in turn were due to an increase in complex I activity in the electron transport chain. Thus, Upf1 helicase deficiency leads to increased mitochondrial complex I activity which produces ROS that signals for cytokine release that drives immune suppression and enhanced tumor growth.

3D Bioprinting of Multicellular Stem Cell‐Derived Constructs to Model Pancreatic Cell Differentiation

AbstractIn vitro models of the pancreas can aid in developing therapies for pancreatic diseases. Nonetheless, most pancreatic tissue engineering is limited to insulin‐secreting β‐cells or pancreatic adenocarcinoma models. Combining all essential tissue components, including exocrine, endocrine, and blood vasculature, is crucial to recapitulate native tissue organization. In this study, extrusion‐based 3D bioprinting to create pancreatic tissue constructs containing both endocrine and exocrine compartments is exploited. Mouse pluripotent stem cell‐derived pancreatic progenitors, pancreatic endothelial cells, and mesenchymal stem cells are bioprinted. During postprinting cultivation, the cells differentiated into exocrine and endocrine lineages, resulting in vascularized pancreatic tissue‐like constructs with multiple cell types. However, the bioprinted constructs contracted significantly postprinting, hindering control of cell positioning and shape preservation. Therefore, 2 strategies to reduce the contraction and deformation of the bioprinted constructs are developed. These bioprinting techniques and biomaterial combinations allow us to investigate the influence of construct design and cellular composition on pancreatic cell fate. The results reveal that increased construct stiffness and endothelial component presence significantly promoted endocrine while suppressing exocrine differentiation. Overall, a novel strategy for pancreatic tissue engineering that advances and holds promise for pancreas disease and development modeling, as well as pharmaceutical testing is demonstrated.

Compressive stress changes the biophysical properties of the nucleus in an in vitro model of pancreatic adenocarcinoma

Compressive stress changes the biophysical properties of the nucleus in an in vitro model of pancreatic adenocarcinoma

Hypoxia-activated prodrug and antiangiogenic therapies cooperatively treat pancreatic cancer but elicit immunosuppressive G-MDSC infiltration.

We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12D TP53+/R172H Pdx1-Cre-derived (KPC-derived) model of pancreatic adenocarcinoma to examine the tumor response and adaptive resistance mechanisms involved in response to 2 established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast with prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8+ T cell response. Bulk tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid-derived suppressor cells (G-MDSCs) through CCL9 secretion. Blockade of the CCL9/CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302, anti-VEGFR-2, and ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.

Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of CEACAM5-expressing cancers

BackgroundT-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA).MethodsWe present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format).ResultsNILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the “LALAPA” mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice.ConclusionsIn summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.

Abstract B125: Neutralizing acidosis with L-DOS47 urease immunoconjugate enhances responses to anti-PD1 checkpoint blockade in a preclinical orthotopic model of pancreatic adenocarcinoma

Abstract Background: Acidosis in the tumor microenvironment is an important immunosuppressive mechanism that leads to tumor growth. We have shown that neutralization of tumor pH using sodium bicarbonate improves responses to immune checkpoint blockade (ICB) in pre-clinical models, but phase I/IIa clinical trials failed due to poor patient compliance. Thus, we aimed to test clinically translatable agents to neutralize tumor acidity and improve cancer outcomes in response to ICB. We tested L-DOS47, a targeted urease immunoconjugate designed to directly neutralize tumor acidity that has been well-tolerated in phase I/IIa trials. The nanobody component of L-DOS47 binds carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6), a cell surface protein highly expressed in gastrointestinal and lung cancers; the urease moiety cleaves endogenous urea into two NH4+ and one CO2 thereby raising local pH. Materials and Methods: Immunohistochemistry screening for CEACAM6 expression and L-DOS47 binding was performed on tumor microarrays from pancreatic ductal adenocarcinoma (PDAC) cases (n=10 plus 2 normal tumor adjacent cases). We established a preclinical in vivo PDAC model by orthotopically inoculating human CEACAM6-expressing KPC961 into immunocompetent mice. Pharmacodynamic studies were performed using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure tumor extracellular pH (pHe) at baseline (pre-L-DOS47) and at various time points post-L-DOS47 (4, 18, 24, 48, 72 and 96 hours). Mean tumor pHe and histograms of pHe distributions were compared among groups. Additional biological replicates were conducted to assess therapeutic efficacy. For these experiments, mice were randomized, after measurable tumors are established, into groups with equal tumor volume averages to initiate twice-weekly therapies with 300 µg anti-PD1 or 90 µg/kg L-DOS47 monotherapies, 300 µg anti-PD1 + 90 µg/kg L-DOS47 compared to a control group receiving no therapy. Tumor volumes were measured weekly by ultrasound imaging up to 4 weeks. Results: Immunohistochemistry screening of patient tumor microarrays confirmed high CEACAM6 expression and L-DOS47 binding to PDAC tissues. CEST-MRI results showed that L-DOS47 increased tumor pHe from 4h to 96h post-injection; however, pHe increases were only evident in tumors with baseline pHe ≤ 6.60. Efficacy studies in the PDAC model showed that the combination of anti-PD1 + L-DOS47 significantly reduced tumor growth when compared with control, L-DOS47 and anti-PD1 groups at week 3 (p<.0001; <.0001 and 0.0368, respectively) and week 4 (p<.0001; <.0001 and 0.0142, respectively). Conclusion: These studies provide strong evidence that L-DOS47, by neutralizing acidic tumor pH, significantly improves responses to anti-PD1 immunotherapy. Citation Format: Bruna V Jardim-Perassi, Pietro Irrera, Dominique Abrahams, Christopher J Whelan, Matthew S Beatty, Samantha R Byrne, Andrew A Odeja, Dario L Longo, Young Ou, Kim Gaspar, Martin Koebel, Shari A Pilon-Thomas, Christof Boehler, Gabrielle M Siegers, Robert J Gillies, Arig A Ibrahim-Hashim. Neutralizing acidosis with L-DOS47 urease immunoconjugate enhances responses to anti-PD1 checkpoint blockade in a preclinical orthotopic model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B125.

Decoding the obesity-cancer connection: lessons from preclinical models of pancreatic adenocarcinoma.

Obesity is a metabolic state of energy excess and a risk factor for over a dozen cancer types. Because of the rising worldwide prevalence of obesity, decoding the mechanisms by which obesity promotes tumor initiation and early progression is a societal imperative and could broadly impact human health. Here, we review results from preclinical models that link obesity to cancer, using pancreatic adenocarcinoma as a paradigmatic example. We discuss how obesity drives cancer development by reprogramming the pretumor or tumor cell and its micro- and macro-environments. Specifically, we describe evidence for (1) altered cellular metabolism, (2) hormone dysregulation, (3) inflammation, and (4) microbial dysbiosis in obesity-driven pancreatic tumorigenesis, denoting variables that confound interpretation of these studies, and highlight remaining gaps in knowledge. Recent advances in preclinical modeling and emerging unbiased analytic approaches will aid in further unraveling the complex link between obesity and cancer, informing novel strategies for prevention, interception, and therapy in pancreatic adenocarcinoma and other obesity-associated cancers.

Trapping all ERBB ligands decreases pancreatic lesions in a murine model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest of cancers. Attempts to develop targeted therapies still need to be established. Some oncogenic mechanisms in PDAC carcinogenesis harness the EGFR/ERBB receptor family. To explore the effects on pancreatic lesions, we attempted simultaneous blockade of all ERBB ligands in a PDAC mouse model. To this end, we engineered a molecular decoy, TRAP-FC , comprising the ligand-binding domains of both EGFR and ERBB4 and able to trap all ERBB ligands. Next, we generated a transgenic mouse model (CBATRAP/0 ) expressing TRAP-FC ubiquitously under the control of the chicken beta-actin promoter and crossed these mice with KRASG12D/+ mice (Kras) to generate Trap/Kras mice. The resulting mice displayed decreased emergence of spontaneous pancreatic lesion areas and exhibited reduced RAS activity and decreased activities of ERBBs, with the exception of ERBB4, which showed increased activity. To identify the involved receptor(s), we employed CRISPR/Cas9 DNA editing to singly delete each ERBB receptor in the human pancreatic carcinoma cell line Panc1. Ablation of each ERBB family member, especially loss of EGFR or ERBB2/HER2, altered signaling downstream of the other three ERBB receptors and decreased cell proliferation, migration and tumor growth. We conclude that simultaneously blocking the entire ERBB receptor family is therapeutically more effective than individually inhibiting only one receptor or ligand in terms of reducing pancreatic tumor burden. In summary, trapping all ERBB ligands can reduce pancreatic lesion area and RAS activity in a murine model of pancreatic adenocarcinoma; hence, it might represent a promising approach to treat PDAC in patients.

Mapping estimates of vascular permeability with a clinical indocyanine green fluorescence imaging system in experimental pancreatic adenocarcinoma tumors.

Pancreatic cancer tumors are known to be avascular, but their neovascular capillaries are still chaotic leaky vessels. Capillary permeability could have significant value for therapy assessment, and its quantification might be possible with macroscopic imaging of indocyanine green (ICG) kinetics in tissue. The capacity of using standard fluorescence surgical systems for ICG kinetic imaging as a probe for capillary leakage was evaluated using a clinical surgical fluorescence imaging system, as interpreted through vascular permeability modeling. Xenograft pancreatic adenocarcinoma models were imaged in mice during bolus injection of ICG to capture the kinetics of uptake. Image analysis included ratiometric data, normalization, and match to theoretical modeling. Kinetic data were converted into the extraction fraction of the capillary leakage. Pancreatic tumors were usually less fluorescent than the surrounding healthy tissues, but still the rate of tumor perfusion could be assessed to quantify capillary extraction. Model simulations showed that flow kinetics stabilized after about 1min beyond the initial bolus injection and that the relative extraction fraction model estimates matched the experimental data of normalized uptake within the tissue. The kinetics in the time period of 1 to 2min post-injection provided optimal differential data between AsPC1 and BxPC3 tumors, although high individual variation exists between tumors. ICG kinetic imaging during the initial leakage phase was diagnostic for quantitative vascular permeability within pancreatic tumors. Methods for autogain correction and normalized model-based interpretation allowed for quantification of extraction fraction and difference identification between tumor types in early timepoints.

The combination of immunotherapy and a glutamine metabolism inhibitor represents an effective therapeutic strategy for advanced and metastatic murine pancreatic adenocarcinoma.

Despite constant advances in cancer research, the treatment of pancreatic adenocarcinoma remains extremely challenging. The intratumoral immunotherapy approach that was developed by our research group and was based on a combination of mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA) showed promising therapeutic effects in various murine tumor models, including a pancreatic adenocarcinoma model (Panc02). However, the efficacy of MBTA therapy in the Panc02 model was negatively correlated with tumor size at the time of therapy initiation. Here, we aimed to further improve the outcome of MBTA therapy in the Panc02 model using the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). The combination of intratumoral MBTA therapy and intraperitoneal administration of DON resulted in the complete elimination of advanced Panc02 subcutaneous tumors (140.8±46.8mm3) in 50% of treated animals and was followed by development of long-term immune memory. In the bilateral Panc02 subcutaneous tumor model, we observed a significant reduction in tumor growth in both tumors as well as prolonged survival of treated animals. The appropriate timing and method of administration of DON were also addressed to maximize its therapeutic effects and minimize its side effects. In summary, our findings demonstrate that the intraperitoneal application of DON significantly improves the efficacy of intratumoral MBTA therapy in both advanced and bilateral Panc02 subcutaneous tumor murine models.

Abstract 5140: Combined administration of poly(I:C) and resiquimod triggers effective antitumoral response in mouse models of pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies worldwide. The lack of clinical symptoms in the early stages of the disease and the poor immunogenicity of PDAC are two major causes of late diagnosis and inefficient therapies. The tumor microenvironment of PDAC is rich in stromal cells inducing desmoplasia, complicating the arrival of medical compounds, as well as in immune cells, that are suppressed by several mediators produced by cancer cells. In this setting, it has been shown that some medications can restore the antitumoral activity of innate immune cells that, in turn, re-activate also adaptive immune cells. In this study, we present the combination of the TLR agonists poly(I:C) (pIC - TLR3) and resiquimod (R848 - TLR7/8) as an immunotherapeutic tool in PDAC. In a subcutaneous model of PDAC (K8484 cells, isolated from KPC mice that spontaneously develop PDAC and recapitulate the features of human pancreatic cancer), the intratumoral administration of pIC+R848 for 5 times every 2-3 days led to the complete regression of the tumor in 100% of the treated mice. Moreover, all mice were protected from a second and a third rechallenge with the same cell line, performed months after the first treatments. Mechanistic studies performed on the tumors with flow cytometry demonstrated an increase of MHC-II positive cells in the monocytic (Ly6C+) compartment, as well as an increase in the number of mature CD8+ T cells and NK cells. Similar findings were demonstrated also in another heterotopic model obtained injecting Panc02 cells, another cell line with a sarcomatous phenotype. Consistently with the flow cytometry data, the depletion of CD4+ and CD8+ T cells abolished the antitumoral efficacy of pIC+R848, while the depletion of NK cells and CSF-1R+ cells was not sufficient to abolish the antitumoral response, suggesting a strong involvement of the adaptive immunity. In line with these findings, the intratumoral administration of pIC+R848 in mice unable to produce IFN-γ failed to reduce the tumor growth. In conclusion, our work clearly demonstrates that pIC+R848 are an effective treatment for PDAC when injected intratumorally and that this activity strongly relies on IFN-γ and adaptive immunity. Further investigations are ongoing to assess their potential in orthotopic models using different routes of administration, in a setting closer to the real clinical situation. Citation Format: Aldo Ummarino, Clément Anfray, Andrea Mariancini, Domenico Supino, Cecilia Garlanda, Fernando Torres Andòn, Alberto Mantovani, Paola Allavena. Combined administration of poly(I:C) and resiquimod triggers effective antitumoral response in mouse models of pancreatic adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5140.

ECM-targeting bacteria enhance chemotherapeutic drug efficacy by lowering IFP in tumor mouse models.

Bacterial cancer therapies aim to manipulate bacteria to effectively deploy therapeutic payloads to tumors. Attenuated bacteria alone often cannot eradicate solid tumors. Attenuated Salmonella can be engineered to deliver cytotoxic drugs to either trigger an immune response or increase antitumor efficacy when combined with chemotherapeutic drugs. However, the extracellular matrix (ECM) surrounding cancer cells forms a barrier that often limits the ability of chemotherapeutic and cytotoxic drugs to penetrate and eliminate tumors. To overcome this limitation, we developed a strategy to combine chemotherapy with an attenuated Salmonella typhimurium strain engineered to secrete HysA protein (from Staphylococcus aureus; Hyaluronidase, HAase) in tumors. The engineered Salmonella effectively degraded hyaluronan (HA), which is a major ECM constituent in tumors, and suppressed tumor growth in mouse models of pancreatic adenocarcinoma (ASPC-1) and breast cancer (4T1). Furthermore, it prolonged survival when combined with chemotherapeutic drugs (doxorubicin or gemcitabine). Upon bacterial colonization, the HAase-mediated ECM degradation decreased interstitial fluid pressure (IFP) in the tumor microenvironment. Additionally, HA degradation using HAase-expressing bacteria in vivo led to decreased binding to the receptor, CD44, expressed in tumors. This may modulate proliferation- and apoptosis-related signal pathways. Therefore, ECM-targeting bacteria can be used as a synergistic anticancer therapeutic agent to maximize chemotherapeutic drug delivery into highly invasive tumors.

Long-circulating magnetoliposomes as surrogates for assessing pancreatic tumour permeability and nanoparticle deposition.

Nanocarriers are candidates for cancer chemotherapy delivery, with growing numbers of clinically-approved nano-liposomal formulations such as Doxil® and Onivyde® (liposomal doxorubicin and irinotecan) providing proof-of-concept. However, their complex biodistribution and the varying susceptibility of individual patient tumours to nanoparticle deposition remains a clinical challenge. Here we describe the preparation, characterisation, and biological evaluation of phospholipidic structures containing solid magnetic cores (SMLs) as an MRI-trackable surrogate that could aid in the clinical development and deployment of nano-liposomal formulations. Through the sequential assembly of size-defined iron oxide nanoparticle clusters with a stabilizing anionic phospholipid inner monolayer and an outer monolayer of independently-selectable composition, SMLs can mimic physiologically a wide range of nano-liposomal carrier compositions. In patient-derived xenograft models of pancreatic adenocarcinoma, similar tumour deposition of SML and their nano-liposomal counterparts of identical bilayer composition was observed in vivo, both at the tissue level (fluorescence intensities of 1.5×108 ± 1.8×107 and 1.2×108 ± 6.3×107, respectively; ns, 99% confidence interval) and non-invasively using MR imaging. We observed superior capabilities of SML as a surrogate for nano-liposomal formulations as compared to other clinically-approved iron oxide nano-formulations (ferumoxytol). In combination with diagnostic and therapeutic imaging tools, SMLs have high clinical translational potential to predict nano-liposomal drug carrier deposition and could assist in stratifying patients into treatment regimens that promote optimal tumour deposition of nanoparticulate chemotherapy carriers. STATEMENT OF SIGNIFICANCE: Solid magnetoliposomes (SMLs) with compositions resembling that of FDA-approved agents such as Doxil® and Onivyde® offer potential application as non-invasive MRI stratification agents to assess extent of tumour deposition of nano-liposomal therapeutics prior to administration. In animals with pancreatic adenocarcinoma (PDAC), SML-PEG exhibited (i) tumour deposition comparable to liposomes of the same composition; (ii) extended circulation times, with continued tumour deposition up to 24 hours post-injection; and (iii) MRI capabilities to determine tumour deposition up to 1 week post-injection, and confirmation of patient-to-patient variation in nanoparticulate deposition in tumours. Hence SMLs with controlled formulation are a step towards non-invasive MRI stratification approaches for patients, enabled by evaluation of the extent of deposition in tumours prior to administration of nano-liposomal therapeutics.

Abstract A079: The quadruplex-binding compound QN-302 in the MIA-PaCa2 pancreatic adenocarcinoma model shows no evidence of cardiac or neurological liabilities at therapeutic doses

Abstract The human genome contains multiple sites of quadruplex (G4) arrangements, with over-representation in promoter regions of many oncogenes. Small molecule G4 stabilization down-regulates the transcription of these genes, leading to selective inhibition of cancer cell growth and anti-tumor activity. We have developed a number of novel G4-binding small molecules. QN-302, the most potent, has significant in vivo activity in animal models for pancreatic cancer and is currently in pre-clinical development with Qualigen Therapeutics. The effects of QN-302 have been examined on (1) a panel of cardiac function receptors, (2) heart rate in vivo, and (3) on the histopathology of organs from treated animals. QN-302 was initially screened in a series of in vitro industry-standard CEREP receptor binding assays. The few apparent indications of significant receptor binding were examined in vivo by means of ELISA assays. Cardiac function was examined using a cardiac heart-beat monitor attached to animals treated with QN-302 and compared to animals dosed with a vehicle control. QN-302 gave negative results in CEREP assays, apart from micromolar inhibition of acetylcholinesterase (ACE) and muscarinic (MUSC) receptors. There was no significant hERG receptor inhibition. There was no significant inhibition of ACE and MUSC receptors in vivo. Heart rate experiments, run at both the proposed therapeutic dose of 1mg/kg and at elevated levels, did not show significant deviations from values for the vehicle control arm. Histopathology of heart, liver and brain tissues from QN-302 treated animals, did not show any deleterious effects from the drug. The lack of effects on ACE and MUSC receptors in vivo, together with the absence of effects on heart, liver and brain tissue from treated animals and the normality of heart rate during treatment, demonstrate that treatment of MIA-Paca2 tumor-bearing mice with QN-302 does not have a cardio-, nephro- or neurotoxic burden at therapeutic doses. QN-302 is bio-available at therapeutic doses and is currently in pre-clinical development with Qualigen Therapeutics Inc Citation Format: Ahmed Ahmed, Tariq Arshad, Stephen Neidle. The quadruplex-binding compound QN-302 in the MIA-PaCa2 pancreatic adenocarcinoma model shows no evidence of cardiac or neurological liabilities at therapeutic doses [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A079.

Establishment of a human 3D pancreatic adenocarcinoma model based on a patient-derived extracellular matrix scaffold

Pancreatic cancer is likely to become one of the leading causes of cancer-related death in many countries within the next decade. Surgery is the potentially curative treatment for pancreatic ductal adenocarcinoma (PDAC), although only 10%–20% of patients have a resectable disease after diagnosis. Despite recent advances in curative surgery the current prognosis ranges from 6% to 10% globally. One of the main issues at the pre-clinical level is the lacking of model which simultaneously reflects the tumour microenvironment (TME) at both structural and cellular levels. Here we describe an innovative tissue engineering approach applied to PDAC starting from decellularized human biopsies in order to generate an organotypic 3D in vitro model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence, mechanical analysis, and scanning electron microscopy. Mass spectrometry confirmed a different extracellular matrix (ECM) composition between decellularized healthy pancreas and PDAC by identifying a total of 110 non-redundant differently expressed proteins. Immunofluorescence analyses after 7 days of scaffold recellularization with PANC-1 and AsPC-1 pancreatic cell lines, were performed to assess the biocompatibility of 3D matrices to sustain engraftment, localization and infiltration. Finally, both PANC-1 and AsPC-1 cells cultured in 3D matrices showed a reduced response to treatment with FOLFIRINOX if compared to conventional bi-dimensional culture. Our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the pancreatic cancer microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of pancreatic cancer response to chemotherapy agents.

Upregulation of dual-specificity phosphatase-26 is required for transforming growth factor β1(TGFβ1)-induced Epithelial-mesenchymal transition in A549 and PANC1 cells.

Transforming Growth Factor β (TGFβ) proteins are potent inducers of the epithelial-mesenchymal transition (EMT) in tumor cells. Although mitogen-activated protein kinase (MAPK) family has been shown to be involved in TGFβ-induced EMT, role of Dual Specificity Phosphatases (DUSP), key regulators of MAPK activity, in TGFβ-induced EMT is largely unkonwn. Real-time qPCR analyses were performed to determine the effect of TGFβ1 on expression of EMT genes and DUSP proteins in the non-small cell lung cancer model A549 and pancreatic adenocarcinoma model PANC1 cells. Western blot analyses were conducted to study the changes in protein levels of EMT proteins and select DUSP proteins, as well as phosphorylations of MAPK proteins upon TGFβ1 stimulation. Small interfering RNA (siRNA) was utilized to reduce expressions of DUSP genes. We observed that the EMT phenotype coincided with increases in phosphorylations of the MAPK proteins ERK1/2, p38MAPK, and JNK upon TGFβ1 stimulation. Real-time qPCR analysis showed increases in DUSP15 and DUSP26 mRNA levels and Western blot analysis confirmed the increase in DUSP26 protein levels in both A549 and PANC1 cells treated with TGFβ1 relative to control. Silencing of DUSP26 expression by siRNA markedly suppressed the effect of TGFβ1 on E-cadherin and mesenchymal genes in the cells. Data provided suggest that TGFβ1 modulates the expression of DUSP genes and that upregulation of DUSP26 may be required for TGFβ1-promoted EMT in A549 and PANC1 cells. Further studies should be carried out to elucidate the requirement of individual DUSPs in TGFβ1-associated EMT in tumor cells.