Pancreatic Tumor Model Research Articles

Relaxin-expressing oncolytic adenovirus induces remodeling of physical and immunological aspects of cold tumor to potentiate PD-1 blockade

BackgroundCurrently, several antibody (Ab)-based therapies have shown excellent therapeutic effects in the clinic. Nonetheless, Ab penetration into tumor tissues is limited due to abnormal vasculature, tumor interstitial pressure, and excessive...

Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder Cancer Cells to Gemcitabine Treatment by Upregulating Deoxycytidine Kinase.

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.

Patient-derived xenograft model of well-differentiated pancreatic neuroendocrine tumor in immunodeficient Balb/c Nude mice

Patient-derived xenograft model of well-differentiated pancreatic neuroendocrine tumor in immunodeficient Balb/c Nude mice

DR5-targeted, chemotherapeutic drug-loaded nanoparticles induce apoptosis and tumor regression in pancreatic cancer in vivo models

Pancreatic cancer is usually advanced and drug resistant at diagnosis. A potential therapeutic approach outlined here uses nanoparticle (NP)-based drug carriers, which have unique properties that enhance intra-tumor drug exposure and reduce systemic toxicity of encapsulated drugs. Here we report that patients whose pancreatic cancers express elevated levels of Death Receptor 5 (DR5) and its downstream regulators/effectors FLIP, Caspase-8, and FADD had particularly poor prognoses. To take advantage of elevated expression of this pathway, we designed drug-loaded NPs with a surface-conjugated αDR5 antibody (AMG 655). Binding and clustering of the DR5 is a prerequisite for efficient apoptosis initiation, and the αDR5-NPs were indeed found to activate apoptosis in multiple pancreatic cancer models, whereas the free antibody did not. The extent of apoptosis induced by αDR5-NPs was enhanced by down-regulating FLIP, a key modulator of death receptor-mediated activation of caspase-8. Moreover, the DNA topoisomerase-1 inhibitor camptothecin (CPT) down-regulated FLIP in pancreatic cancer models and enhanced apoptosis induced by αDR5-NPs. CPT-loaded αDR5-NPs significantly increased apoptosis and decreased cell viability in vitro in a caspase-8- and FADD-dependent manner consistent with their expected mechanism-of-action. Importantly, CPT-loaded αDR5-NPs markedly reduced tumor growth rates in vivo in established pancreatic tumor models, inducing regressions in one model. These proof-of-concept studies indicate that αDR5-NPs loaded with agents that downregulate or inhibit FLIP are promising candidate agents for the treatment of pancreatic cancer.

A novel pancreatic tumour and stellate cell 3D co-culture spheroid model

BackgroundPancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells.MethodA spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting.ResultsWe found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids.ConclusionsWe developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

Preclinical study of oncolytic adenovirus-mediated cytotoxic gene therapy with gemcitabine for chemoresistance of desmoplastic tumor.

e15507 Background: Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. Methods: To overcome the limitations of various conventional therapeutics against desmoplastic pancreatic cancer, we utilized an oncolytic adenovirus (oAd) for combination therapy with gemcitabine, a standard chemotherapeutic for pancreatic cancer treatment. Here, we report the results of preclinical pharmacology and efficology studies conducted in pancreatic cancer model. Results: Efficacy studies in orthotopic pancreatic xenograft tumor model demonstrated that oAd effectively attenuated tumor cell proliferation. oAd further prevented metastasis of pancreatic cancer and sensitized pancreatic tumor to gemcitabine treatment. Furthermore, oAd augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived pancreatic tumor spheroids. These results suggest that oAd enhance the therapeutic effect of chemotherapeutic agent in chemo-resistant and desmoplastic pancreatic tumor, thus overcoming the preexisting limitations of standard treatments. Conclusions: Overall, these results demonstrate that local administration of an oncolytic adenovirus with gemcitabine is well tolerated and support moving this investigational approach into human trials.

Ultrasound-Activated Cascade Effect for Synergistic Orthotopic Pancreatic Cancer Therapy.

SummaryIn some malignant tumor, especially for pancreatic tumor, poor solid-tumor penetration of nanotherapeutics impedes their treatment efficacy. Herein, we develop a polymer-peptide conjugate with the deep tissue penetration ability, which undergoes a cascade process under ultrasound (US), including (1) the singlet oxygen 1O2 is generated by P18, (2) the thioketal bond is cleaved by the 1O2, (3) the departure of PEG chains leads to the in situ self-assembly, and (4) the resultant self-assembled PK nanoparticles show considerable cellular internalization. Owing to the synergistic effect of US on increasing the membrane permeability, the endocytosis and lysosome escape of PK nanoparticles are further enhanced effectively, resulting in the improved therapeutic efficacy. Thanks to the high tissue-penetrating depth and spatial precision of US, PTPK presents enhanced tumor inhibition in an orthotopic pancreatic tumor model. Therefore, the US-activated cascade effect offers a novel perspective for precision medicine and disease theranostics.

Abstract IA01: Deconstructing K-RAS oncogenic signaling in lung and pancreatic tumors

Abstract The presence of K-RAS oncogenes in human tumors has been known for over 35 years. Indeed, extensive analysis of cancer genomes has revealed that they are present in at least a quarter of all solid tumors. Moreover, there is overwhelming evidence that they represent the initiating event in lung and pancreatic adenocarcinomas, two cancer types with some of the worse prognoses. Recent studies in genetically engineered mouse (GEM) tumor models have shown that inhibition of K-RAS oncogene expression results in rapid tumor regression. Yet in spite of recent progress, K-RAS oncogenes remain for the most part undruggable. Ironically, the main K-RAS downstream effectors are druggable kinases. Selective inhibitors against members of the MAPK and PI3K signaling pathways have been developed over the last two decades. Yet none of them has yielded suitable results in clinical trials to merit FDA approval against K-RAS mutant malignancies. In an effort to identify K-RAS vulnerabilities that may have been missed using biochemical or pharmacologic approaches, we decided a decade ago to deconstruct K-Ras oncogenic signaling in GEM tumor models by interrogating the effect of ablating or inactivating its downstream kinases during tumor initiation. In addition, we examined the consequences of systemically eliminating or inhibiting these targets during adult homeostasis, in order to evaluate the potential toxic effects that may result from tampering with the MAPK and PI3K signaling pathways. These studies revealed three classes of downstream targets: those whose ablation had no consequences on tumor development, those that prevented tumor development but were unacceptably toxic when ablated systemically, and a limited number (c-Raf and Cdk4) that prevented or thwarted tumor development without causing significant toxicities upon systemic elimination. In addition to these downstream targets, we were also able to identify the EGFR as a potential target in pancreatic but not in lung tumors. More recently, we have established a new generation of “therapeutic” GEM tumor models in which we can separate, both spatially and temporally, tumor development from target ablation/inactivation by using two independent recombinase systems. In these models, whereas K-Ras or K-Ras/TP53-driven tumor development is mediated by the Flp(o)/frt system, we use the inducible CreERT2/loxP system for systemic target ablation/inactivation. These “therapeutic” models have allowed us to explore the therapeutic potential of these previously validated targets in tumor-bearing mice. Our results have revealed that c-Raf ablation induces significant levels of tumor regression in K-Ras/TP53-driven lung tumors, a therapeutic effect that is further potentiated by the concomitant expression of a kinase dead isoform of Cdk4. In the corresponding GEM pancreatic tumor model, ablation or inactivation of c-Raf, Cdk4, or EGFR failed to prevent tumor regression, although tampering with Cdk4 or EGFR resulted in significant tumor delays. Importantly, concomitant ablation of c-Raf and EGFR in K-Ras/TP53-driven advanced pancreatic tumors resulted in complete tumor regression in half of the mice. Preliminary comparison of these “responder” tumors with those that did not respond to c-Raf/EGFR ablation is revealing some vulnerabilities that may provide additional targets against these advanced tumors. We hope that these results may serve to pave the way to define more efficacious therapeutic strategies against K-RAS mutant tumors that might eventually be translated to the clinic. Citation Format: Mariano Barbacid. Deconstructing K-RAS oncogenic signaling in lung and pancreatic tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA01.

Organoid-Transplant Model Systems to Study the Effects of Obesity on the Pancreatic Carcinogenesis in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality among adults in developed countries. The discovery of the most common genetic alterations as well as the development of organoid models of pancreatic cancer have provided insight into the fundamental pathways driving tumor progression from a normal cell to non-invasive precursor lesion and finally to widely metastatic disease, offering new opportunities for identifying the key driver of cancer evolution. Obesity is one of the most serious public health challenges of the 21st century. Several epidemiological studies have shown the positive association between obesity and cancer-related morbidity/mortality, as well as poorer prognosis and treatment outcome. Despite strong evidence indicates a link between obesity and cancer incidence, the molecular basis of the initiating events remains largely elusive. This is mainly due to the lack of an accurate and reliable model of pancreatic carcinogenesis that mimics human obesity-associated PDAC, making data interpretation difficult and often confusing. Here we propose a feasible and manageable organoid-based preclinical tool to study the effects of obesity on pancreatic carcinogenesis. Therefore, we tracked the effects of obesity on the natural evolution of PDAC in a genetically defined transplantable model of the syngeneic murine pancreatic preneoplastic lesion (mP) and tumor (mT) derived-organoids that recapitulates the progression of human disease from early preinvasive lesions to metastatic disease. Our results suggest that organoid-derived transplant in obese mice represents a suitable system to study early steps of pancreatic carcinogenesis and supports the hypothesis that inflammation induced by obesity stimulates tumor progression and metastatization during pancreatic carcinogenesis.

Novel Non-integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models

We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.

Squamous trans-differentiation of pancreatic cancer cells promotes stromal inflammation.

A highly aggressive subset of pancreatic ductal adenocarcinomas undergo trans-differentiation into the squamous lineage during disease progression. Here, we investigated whether squamous trans-differentiation of human and mouse pancreatic cancer cells can influence the phenotype of non-neoplastic cells in the tumor microenvironment. Conditioned media experiments revealed that squamous pancreatic cancer cells secrete factors that recruit neutrophils and convert pancreatic stellate cells into cancer-associated fibroblasts (CAFs) that express inflammatory cytokines at high levels. We use gain- and loss-of-function approaches to show that squamous-subtype pancreatic tumor models become enriched with neutrophils and inflammatory CAFs in a p63-dependent manner. These effects occur, at least in part, through p63-mediated activation of enhancers at pro-inflammatory cytokine loci, which includes IL1A and CXCL1 as key targets. Taken together, our findings reveal enhanced tissue inflammation as a consequence of squamous trans-differentiation in pancreatic cancer, thus highlighting an instructive role of tumor cell lineage in reprogramming the stromal microenvironment.

Supramolecular Aggregation-Induced Emission Nanodots with Programmed Tumor Microenvironment Responsiveness for Image-Guided Orthotopic Pancreatic Cancer Therapy

Supramolecular nanomaterials as drug carriers have recently received increasing attention due to their intrinsic merits such as high stability, strong inclusion capability, and facile modification of the parental structure; however, intelligent ones with combined capacities of long blood circulation, highly efficient tumor cell uptake, and site-oriented drug release inside tumor cells are still rather limited. Herein, we report a strategy using supramolecular aggregation-induced emission (AIE) nanodots for image-guided drug delivery, which integrate both the advantages of AIE and supramolecular nanomaterials. The supramolecular AIE dots are prepared by the host-guest coassembly of the matrix metalloproteinase-2 (MMP-2) sensitive PEG-peptide (PEG2000-RRRRRRRR (R8)-PLGLAG-EKEKEKEKEKEK (EK6)) and functional α-cyclodextrins (α-CD) derivatives that are conjugated with the anticancer drug gemcitabine (GEM) and a far-red/near-infrared fluorescent rhodanine-3-acetic acid-based AIE luminogen, respectively. The supramolecular AIE dots realize long blood circulation time by virtue of the zwitterionic stealth peptide EK6. After largely accumulating in tumor tissues by the enhanced permeability and retention effect, the supramolecular AIE dots can successively respond to the tumor-overexpressed MMP-2 and intracellular reductive microenvironment, achieving both enhanced cancer cellular uptake and selective GEM release within cancer cells, which thus exhibit excellent tumor inhibition ability in both subcutaneous and orthotopic pancreatic tumor models.

Concurrent Injection of Unlabeled Antibodies Allows Positron Emission Tomography Imaging of Programmed Cell Death Ligand 1 Expression in an Orthotopic Pancreatic Tumor Model.

Purpose: Among the treatment options for pancreatic ductal adenocarcinoma (PDAC) are antibodies against the programmed cell death receptor 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway. Positron emission tomography (PET) has been successfully used to assess PD-1/PD-L1 signaling in subcutaneous tumor models, but orthotopic tumor models are increasingly being recognized as a better option to accurately recapitulate human disease. However, when PET radiotracers have high uptake in the liver and spleen, it can obscure signals from the adjacent pancreas, making visualization of the response in orthotopic pancreatic tumors technically challenging. In this study, we first investigated the impact of radioisotope chelators on the biodistribution of 64Cu-labeled anti-PD-1 and anti-PD-L1 antibodies and compared the distribution profiles of anti-PD-1 and anti-PD-L1 antibodies. We then tested the hypothesis that co-injection of unlabeled antibodies reduces uptake of 64Cu-labeled anti-PD-L1 antibodies in the spleen and thereby permits accurate delineation of orthotopic pancreatic tumors in mice. Procedures: We established subcutaneous and orthotopic mouse models of PDAC using KRAS* murine pancreatic cancer cells with a doxycycline-inducible mutation of KRASG12D. We then (1) compared the biodistribution of 64Cu-labeled anti-PD-1 with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) and 2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) used as the chelators in the orthotopic model; (2) compared the biodistribution of [64Cu]Cu-NOTA-anti-PD-1 and [64Cu]Cu-NOTA-anti-PD-L1 in the orthotopic model; and (3) imaged subcutaneous and orthotopic KRAS* tumors with [64Cu]Cu-NOTA-anti-PD-L1 with and without co-injection of unlabeled anti-PD-L1 as the blocking agent. Results: [64Cu]Cu-NOTA-anti-PD-L1 was a promising imaging probe. By co-injection of an excess of unlabeled anti-PD-L1, background signals of [64Cu]Cu-NOTA-anti-PD-L1 from the spleen were significantly reduced, leading to a clear delineation of orthotopic pancreatic tumors. Conclusions: Co-injection with unlabeled anti-PD-L1 is a useful method for PET imaging of PD-L1 expression in orthotopic pancreatic cancer models.

Abstract A81: Hyaluronan-dependent skewing of tumor-associated macrophage (TAM) phenotype in a murine pancreatic tumor model is associated with increased tumor hypoxia

Abstract Hyaluronan (HA) is a naturally occurring glycosaminoglycan that accumulates in the tumor microenvironment of several cancer types, including pancreatic ductal adenocarcinoma (PDA), where it is an independent negative predictor of survival. An ongoing phase 3 study is evaluating pegvorhyaluronidase alfa (PEGPH20; PVHA), a first-in-class biologic that enzymatically degrades HA in the tumor microenvironment, in combination with gemcitabine and nab-paclitaxel in metastatic PDA. Recently, we reported that HA-accumulation can promote tumor hypoxia; however, the effects this may have on tumor-infiltrating immune cells are unknown. Transduction of syngeneic murine PDA cells (Pan02) with hyaluronan synthase 3 (HAS3) generates tumors (Pan02-HAS3) containing high levels of HA, and a higher frequency of TAMs with an immunosuppressive phenotype (CD206-high MHCII-low), relative to tumors generated by empty vector-transduced cells (Pan02-EV). Given the association of this TAM phenotype with tumor hypoxia, we assessed the relationship between tumor hypoxia and the TAM phenotypes of Pan02-HAS3 and Pan02-EV tumors. To confirm whether the effects seen in Pan02-HAS3 tumors require HA synthesis, we also generated tumors expressing an enzymatically inactive HAS3 mutant (Pan02-HAS3 D215A). Tumor-bearing mice were injected with pimonidazole hydrochloride 2 hours prior to harvest and dissociation of tumor tissue; subsequent flow cytometric analysis of tumor-infiltrating immune cells included an antibody against pimonidazole hydrochloride adducts (HP-1) to detect cells residing in hypoxic zones. A greater mean percentage of TAMs isolated from Pan02-HAS3 peritibial tumors had high levels of HP-1 staining (HP-1+) compared with TAMs isolated from either Pan02-EV or Pan02-HAS3 D215A tumors (18.2%, 4.2% and 3.9%, respectively). Furthermore, HP-1+ cells were enriched among CD206-high MHCII-low TAMs compared with their MHCII-high counterparts in Pan02-HAS3 tumors (38.7% vs 11.9%). Likewise, more HP-1+ cells were seen among dendritic cells (DCs) isolated from Pan02-HAS3 tumors compared with either Pan02-EV or Pan02-HAS3 D215A tumors (6.9%, 1.3% and 0.8%, respectively). Similar findings were obtained in orthotopic tumors. In contrast, no consistent differences in the percentages of HP-1+ CD8 T cells or NK cells were observed across the three tumor types in this study. Finally, exposure of cultured macrophages to hypoxia recapitulated some of the changes in gene expression observed in TAMs sorted from Pan02-HAS3 tumors, including increased expression of Arg1 and Nos2. These data suggest that tumor HA accumulation may promote an immunosuppressive TAM phenotype by creating tumor hypoxic zones that are subsequently infiltrated by TAMs, but not T or NK cells. Citation Format: Trevor Kimbler, Jessica Cowell, Jisook Lee, Benjamin J. Thompson. Hyaluronan-dependent skewing of tumor-associated macrophage (TAM) phenotype in a murine pancreatic tumor model is associated with increased tumor hypoxia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A81.

Abstract A46: Degradation of hyaluronan from the tumor extracellular matrix increases infiltration of CD8+ T cells and natural killer cells in an orthotopic pancreatic tumor model

Abstract T-cell migration and infiltration in solid tumors is a critical step for maximizing the efficacy of many cancer immunotherapies. Hyaluronan (HA) is a glycosaminoglycan that accumulates in the extracellular matrix (ECM) of a range of solid tumors, including pancreatic ductal adenocarcinoma (PDA). PEGylated recombinant human hyaluronidase PH20, pegvorhyaluronidase alfa (PEGPH20; PVHA), is a novel, first-in-class biologic that enzymatically degrades HA in the tumor microenvironment (TME) and is in clinical development in combination with chemotherapy for the treatment of HA-accumulating tumors. Previously, in a murine PDA tumor model, we demonstrated that accumulation of HA restricts T-cell infiltration and promotes immunosuppression. In the present study, we hypothesized that degradation of HA by PEGPH20 would increase access of CD8+ T cells to the tumor. To test this hypothesis, Pan02 murine PDA cells were engineered to overexpress hyaluronan synthase 3 (Pan02-HAS3) and were implanted orthotopically into the pancreas. In Pan02-HAS3 tumors, HA accumulation was 53-fold higher (2606 ng/mg) compared with Pan02 parental tumors (50 ng/mg) as determined by HA ELISA. Degradation of HA in Pan02-HAS3 tumors with intravenous PEGPH20 as a single agent inhibited tumor growth by 58% at a dose of 0.0375 mg/kg and 73% at a dose of 1 mg/kg. Flow cytometric analysis of tumor-infiltrating immune cells in PEGPH20-treated tumors demonstrated a significant increase in the numbers of cytotoxic CD8+ tumor-infiltrating lymphocytes and natural killer (NK) cells versus control tumors. However, no changes in CD8+ T-cell proliferation were detected, as measured by Ki67 expression or bromodeoxyuridine (BrdU) incorporation. Degradation of HA in the TME was also associated with an increased frequency of CD103+ dendritic cells and a decreased frequency of immunosuppressive CD206+ tumor-associated macrophages. These data support prior reports that accumulation of HA in the TME restricts T-cell infiltration and promotes immunosuppression, and that PEGPH20 treatment may facilitate access of CD8+ T cells and NK cells into tumors. These findings are consistent with prior studies and support the ongoing clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors to improve antitumor efficacy in HA-accumulating tumors. Citation Format: Jisook Lee, Trevor Kimbler, Curtis Thompson, Benjamin J. Thompson. Degradation of hyaluronan from the tumor extracellular matrix increases infiltration of CD8+ T cells and natural killer cells in an orthotopic pancreatic tumor model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A46.

In vivo bioluminescence tomography-guided radiation research platform for pancreatic cancer: an initial study using subcutaneous and orthotopic pancreatic tumor models.

Genetically engineered mouse model(GEMM) that develops pancreatic ductal adenocarcinoma(PDAC) offers an experimental system to advance our understanding of radiotherapy(RT) for pancreatic cancer. Cone beam CT(CBCT)-guided small animal radiation research platform(SARRP) has been developed to mimic the RT used for human. However, we recognized that CBCT is inadequate to localize the PDAC growing in low image contrast environment. We innovated bioluminescence tomography(BLT) to guide SARRP irradiation for in vivo PDAC. Before working on the complex PDAC-GEMM, we first validated our BLT target localization using subcutaneous and orthotopic pancreatic tumor models. Our BLT process involves the animal transport between the BLT system and SARRP. We inserted a titanium wire into the orthotopic tumor as the fiducial marker to track the tumor location and to validate the BLT reconstruction accuracy. Our data shows that with careful animal handling, minimum disturbance for target position was introduced during our BLT imaging procedure(<0.5mm). However, from longitudinal 2D bioluminescence image(BLI) study, the day-to-day location variation for an abdominal tumor can be significant. We also showed that the 2D BLI in single projection setting cannot accurately capture the abdominal tumor location. It renders that 3D BLT with multiple-projection is needed to quantify the tumor volume and location for precise radiation research. Our initial results show the BLT can retrieve the location at 2mm accuracy for both tumor models, and the tumor volume can be delineated within 25% accuracy. The study for the subcutaneous and orthotopic models will provide us valuable knowledge for BLT-guided PDAC-GEMM radiation research.

Polymeric modification of gemcitabine via cyclic acetal linkage for enhanced anticancer potency with negligible side effects

Gemcitabine (GEM) is a powerful anticancer drug for various cancers. However, the anticancer efficacy and the side effects should be addressed for effective therapeutics. To this end, we created a GEM-conjugated polymer (P-GEM) based on cyclic acetal linkage as a delivery carrier of GEM. The obtained P-GEM stably conjugated GEM at physiological pH (i.e., bloodstream), but released GEM in response to acidic environments such as endosome/lysosome. After systemic administration of P-GEM for mice bearing subcutaneous tumors, it achieved prolonged blood circulation and enhanced tumor accumulation relative to free GEM system. In addition, the polymer-drug conjugate structure of P-GEM realized effective distribution in the tumor tissues toward the induction of apoptosis in most areas of the tumor sites. Of note, the molecular design of P-GEM achieved minimal accumulation in normal tissues, resulting in negligible GEM-derived adverse effects (e.g., gastrointestinal toxicity and hematotoxicity). Ultimately, even four times smaller dose of P-GEM on a GEM basis realized comparable/higher tumor growth suppression effect for two distinct pancreatic tumor models, compared to free GEM system. The obtained results suggest the huge potential of the present design of GEM-conjugated polymer for anticancer therapeutics.

Effects of evodiamine on PI3K/Akt and MAPK/ERK signaling pathways in pancreatic cancer cells.

The effective antitumor drug evodiamine (EVO) is attracting increased attention. Therefore, the present study aimed to investigate the effects of EVO on the proliferation, apoptosis and autophagy of human pancreatic cancer (PC) cell lines in vitro and in vivo. Human PANC-1 and SW1990 PC cell lines were treated with different concentrations of EVO and proliferation was detected using a Cell Counting Kit (CCK)-8 assay. Colony formation and wound-healing assays showed that EVO inhibited PC cell viability and migration, and apoptosis was detected using flow cytometry. Western blotting and immunofluorescence detected the expression of proteins in PANC-1 and SW1990 cells. The PANC-1 cells were used to establish an orthotopic pancreatic tumor model in nude mice. Tumor-bearing nude mice were administered with different concentrations of EVO, and growth was monitored. High-resolution positron emission tomography and fluorine-18-labeled fluorodeoxyglucose were used to monitor the tumor/non-tumor (T/NT) ratio and standard uptake value (SUV) of the mice, which were subsequently sacrificed to measure the transplanted tumor weight. Apoptosis increased with increasing EVO concentration. The EVO-treated PC cells exhibited significantly higher expression of LC3II than the controls cells. EVO decreased LC3II, enhanced P62 and inhibited the expression of Akt, extracellular-signal-regulated protein kinase (ERK)1/2 and p38. Compared with the control group, the T/NT ratio, SUV and tumor weight decreased more markedly in the EVO-treated group. The tumor expression of phosphorylated AKT, detected using immunohistochemistry, decreased with increasing EVO doses in vivo. EVO induced PC cell apoptosis by inhibiting phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase/ERK and inhibiting the phosphorylation of signal transducer and activator of transcription activator 3 in PC cells to inhibit autophagy, suggesting that EVO may be considered as a novel PC treatment.

Modulation of Extracellular Matrix Rigidity Via Riboflavin-mediated Photocrosslinking Regulates Invasive Motility and Treatment Response in a 3D Pancreatic Tumor Model.

In this study, we evaluate the use of riboflavin-mediated collagen photocrosslinking as an experimental tool to modulate extracellular matrix (ECM) mechanical properties in 3D in vitro tumor models. Using this approach in conjunction with 3D pancreatic tumor spheroid transplants, we show that the extent of matrix photocrosslinking in reconstituted hydrogels with fixed protein concentration scales inversely with the extent of invasive progression achieved by cells infiltrating into the surrounding ECM from primary transplanted spheroids. Using cross-linking to manipulate the extent of invasion into ECM in conjunction with imaging-based treatment assessment, we further leverage this approach as a means for assaying differential therapeutic response in primary nodule and ECM-invading populations and compare response to verteporfin-based photodynamic therapy (PDT) and oxaliplatin chemotherapy. Treatment response data shows that invading cell populations (which also exhibit markers of increased EMT) are highly chemoresistant yet have significantly increased sensitivity to PDT relative to the primary nodule. In contrast, the oxaliplatin treatment achieves greater growth inhibition of the primary nodule. These findings may be significant in themselves, while the methodology developed here could have a broader range of applications in developing strategies to target invasive disease and/or mecahanobiological determinants of therapeutic response in solid tumors.

Construction of Multi-Scale Model of Pancreatic Cancer Tumor from MRI Image and Pathological Microscopic Images

Construction of Multi-Scale Model of Pancreatic Cancer Tumor from MRI Image and Pathological Microscopic Images