Patient-derived Pancreatic Cancer Xenograft Models Research Articles

Abstract 3121: Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers

Abstract To address the need for targeted therapy in the treatment of HER3-positive solid tumors, the human HER3 mAb, seribantumab, was evaluated in the context of an antibody-drug conjugate (ADC). As a proof-of-concept, seribantumab was conjugated with a cleavable valine-citrulline linker and monomethyl auristatin E (MMAE) payload via the stochastic cysteine conjugation method to yield HER3-ADC1. A drug-antibody ratio of 4 was selected for preclinical assessment in HER3-expressing models. Methods: HER3-ADC1 was evaluated in vitro and in vivo, with patritumab deruxtecan (patri-DXd) as a comparator. Binding to BT474 breast carcinoma cells (HER3 high; immunohistochemical (IHC) staining intensity 3+) was measured by flow cytometry. Internalization properties were established using seribantumab and patritumab coupled to Fab-AF488 following 4 hours incubation in SK-BR-3 breast adenocarcinoma (HER3 high; IHC 3+) and HCC1569 breast carcinoma (HER3 low; IHC 0-1+) cell lines. In vitro cytotoxicity was evaluated for HER3-ADC1, isotype-MMAE and free MMAE payload as well as patri-DXd, isotype-DXd and free deruxtecan payload in BT474 cells, SK-BR-3 cells and NCI-H446 lung carcinoma (HER3 low; IHC 0-1+) cells. In vivo anti-tumor activity was assessed for HER3-ADC1, isotype-MMAE, patri-DXd and isotype-DXd in patient derived xenograft (PDX) models of pancreatic (HER3 high; IHC 3+) and breast cancer (HER3 low; IHC 0-1+). Results: HER3-ADC1 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on HER3 expression. In assays investigating antibody internalization, seribantumab displayed similar internalization capacity as patritumab. For both HER3 mAbs, internalization was greater in a HER3 high than a HER3 low cell line. In cytotoxicity assays, HER3-ADC1 and patri-DXd displayed HER3 expression level-dependent cell killing. HER3-ADC1 outperformed patri-DXd in HER3 high cell lines based on in vitro cytotoxicity. HER3-ADC1 and patri-DXd were ineffective against NCI-H466 cells with low HER3 expression. In a PDX model of pancreatic cancer (HER3 high), HER3-ADC1 induced tumor regression, while isotype-MMAE had only a moderate anti-tumor effect. Both patri-DXd and isotype-DXd had moderate anti-tumor effects, indicating a lack of HER3 target dependence. In a PDX model of breast cancer (HER3 low), HER3-ADC1 and isotype-MMAE lacked anti-tumor activity; isotype-DXd had a moderate anti-tumor effect and patri-DXd had a substantial anti-tumor effect. Conclusions: HER3-ADC1 demonstrated target-dependent in vitro cytotoxicity and in vivo anti-tumor activity in a HER3-expressing pancreatic cancer PDX model. Results from in vitro and in vivo studies highlight the promising therapeutic potential of a seribantumab-based ADC for patients with HER3-expressing cancers. Additional results on the optimization and characterization of HER3-ADC1 will be presented. Citation Format: Thomas O'Hare, Jaclyn Cleveland, Valerie M. Jansen, David Dornan. Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3121.

Abstract A091: The RAF/MEK clamp avutometinib as the backbone of therapy for pancreatic cancer: Novel combinations with standard of care chemotherapy, FAK inhibitors, KRAS G12D inhibitors and/or autophagy inhibitors

Abstract KRAS mutations (mt) occur in up to 98% of pancreatic ductal adenocarcinoma (PDAC) and represent a key initiating event in PDAC carcinogenesis. Therapeutic efforts targeting the RAS/RAF/MEK/ERK (MAPK) pathway with MEK-only inhibitors have been unsuccessful in substantially modifying PDAC prognosis. Thus, novel strategies are needed to overcome putative mechanisms of resistance to MEK inhibition such as focal adhesion kinase (FAK) pathway activation. Another hallmark of PDAC is its high stromal density, which is thought to limit the penetration of cytotoxic drugs and T cells into the tumor and has been shown to be correlated with FAK hyperactivation, altogether supporting co-targeting the MAPK and FAK pathways to achieve deep and durable responses for patients with PDAC. Avutometinib is a RAF/MEK clamp that potently inhibits MEK kinase activity and induces dominant negative RAF-MEK complexes preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows avutometinib to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEK-only inhibitors. Preclinically, avutometinib has shown strong anti-proliferative potency across tumor cell lines carrying KRAS mt including PDAC cell lines. Clinically, avutometinib in combination with the FAK inhibitor (FAKi) defactinib received breakthrough therapy designation for the treatment of patients with recurrent low-grade serous ovarian cancer. Here, we tested the combination of avutometinib and FAK inhibition with standard of care chemotherapy in PDAC mouse models. In a KRAS/p53 pancreatic cancer mouse model, we found that whereas the combination of avutometinib + FAKi induced tumor inhibition and increased survival, addition of chemotherapy (gemcitabine + paclitaxel as a surrogate for nab-paclitaxel) to avutometinib/FAKi induced tumor regression. These results support the ongoing clinical study of avutometinib and defactinib in combination with standard of care chemotherapy (gemcitabine/nab-paclitaxel) for patients with front-line metastatic PDAC (NCT05669482). Other combinations being explored preclinically include avutometinib with G12D inhibition (G12Di) in KRAS G12D PDAC, which accounts for 28% of patients with PDAC. In 3D culture, avutometinib was synergistic with the G12Di MRTX1133 in reducing viability of a panel of KRAS G12D PDAC cell lines. In mouse models, avutometinib enhanced anti-tumor efficacy of MRTX1133 in a KRAS G12D pancreatic cancer patient-derived xenograft model. Furthermore, in KRAS mt PDAC cell lines, avutometinib was synergistic with the autophagy inhibitors chloroquine or apilimod. Indeed, dual treatment with avutometinib + MRTX1333 synergistically decreased proliferation and increased autophagic flux in KRAS G12D PDAC cell lines, supporting testing the triple combination of avutometinib + G12Di + autophagy inhibitor in patients with KRAS G12D pancreatic cancers. In conclusion, we show that avutometinib is a unique RAF/MEK clamp with the potential to become the backbone of therapy for pancreatic cancers. Citation Format: Silvia Coma, Xiuting Liu, Noah L. Pieper, Wen-Hsuan Chang, Kirsten L. Bryant, Channing J. Der, David G. DeNardo, Jonathan A. Pachter. The RAF/MEK clamp avutometinib as the backbone of therapy for pancreatic cancer: Novel combinations with standard of care chemotherapy, FAK inhibitors, KRAS G12D inhibitors and/or autophagy inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A091.

Structure-Activity Relationship of FL118 Platform Position 7 Versus Position 9-Derived Compounds and Their Mechanism of Action and Antitumor Activity.

Structurally, FL118 is a camptothecin analogue and possesses exceptional antitumor efficacy against human cancer through a novel mechanism of action (MOA). In this report, we have synthesized and characterized 24 FL118 Position 7-substituted and 24 FL118 Position 9-substituted derivatives. The top compounds were further characterized for their MOA in colorectal cancer (CRC) models using CRC patient-derived xenograft (PDX) models and pancreatic cancer PDX models to evaluate their antitumor activities. Four FL118 Position 7-substituted derivatives showed significantly better antitumor efficacy than the FL118 Position 9-substituted derivatives. The four identified compounds also appeared to have better antitumor activity than their parental platform FL118. Interestingly, RNA-Seq analyses indicated that three of the four compounds exerted antitumor effects via an MOA similar to FL118, which provided an intriguing opportunity for follow-up studies. Extended in vivo studies revealed that FL77-6 (7-(4-ethylphenyl)-FL118), FL77-9 (7-(4-methoxylphenyl)-FL118), and FL77-24 (7-(3, 5-dimethoxyphenyl)-FL118) exhibit potential for further development toward clinical trials.

MP04-13 IMMUNOPHENOTYPE ANALYSIS OF TUMOR-INFILTRATING IMMUNE CELLS TO ELUCIDATE THE MECHANISM OF ANTITUMOR EFFECT OF IL-7 AND CCL19 PRODUCING CAR-T CELLS AGAINST SOLID CANCER

You have accessJournal of UrologyCME1 Apr 2023MP04-13 IMMUNOPHENOTYPE ANALYSIS OF TUMOR-INFILTRATING IMMUNE CELLS TO ELUCIDATE THE MECHANISM OF ANTITUMOR EFFECT OF IL-7 AND CCL19 PRODUCING CAR-T CELLS AGAINST SOLID CANCER Shunsuke Goto, Koji Tamada, and Masatoshi Eto Shunsuke GotoShunsuke Goto More articles by this author , Koji TamadaKoji Tamada More articles by this author , and Masatoshi EtoMasatoshi Eto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chimeric antigen receptor (CAR)-T cell therapy against solid cancer is challenging. We reported that mouse CAR-T cells modified to produce interleukin- (IL)-7 and CCL19 (7×19 CAR-T) enhance the therapeutic efficacy against solid tumors by recruiting not only administered CAR-T cells but also endogenous T cells and DCs into tumors and inducing long-term antitumor memory responses. Furthermore, we reported that human 7×19 CAR-T cells show enhanced antitumor efficacy against patient-derived xenograft (PDX) tumor expressing mesothelin, for which 10% of renal cell carcinoma are positive. In this research, we explore the mechanism of antitumor activity of 7×19 CAR-T cells and object to prevail the difference between conventional CAR-T cells (Conv.) and 7×19, especially focusing on the immunophenotype of tumor-infiltrating lymphocytes (TILs). METHODS: We produced a novel anti-human mesothelin 7×19 CAR-T cells, then assessed the antitumor activity against solid tumor in vivo using a malignant mesothelioma orthotopic tumor and a pancreatic cancer PDX models. Tumor was resected after the administration of Conv. or 7×19 CAR-T cells and analyzed TILs by flowcytometry focusing on the number, exhaustion marker, phenotype and cytokine producing ability. RESULTS: 7×19 CAR-T cells showed superior antitumor efficacy compared to Conv. CAR-T cells in the orthotopic and PDX tumor models. In the PDX treatment model, TIL analysis revealed that not only CAR-T cells but also non-CAR-T cells were infiltrated effectively into tumors by 7×19 treatment compared to Conv. Exhaustion marker expressions such as PD-1 and TIGIT were significantly downregulated in 7×19 than Conv. The examination of the ability of TILs to express cytokines and effector molecules showed that significant increases of TNF-α and Granzyme B, as well as a trend of increased IFN-γ in 7×19 CAR-T cells. The analysis of splenocyte after the treatment revealed that 7×19 CAR-T cells maintained their effector memory phenotype. CONCLUSIONS: It was found that 7×19 CAR-T cells showed enhanced antitumor potential by the maintenance of their effector functions. These data support the foresight of this therapy as a treatment option for patients with mesothelin-positive renal cell carcinoma. Source of Funding: Practical Research for Innovative Cancer Control, and Project for Cancer Research and Therapeutic Evolution (P-CREATE) 16770206 by Japan Agency for Medical Research and Development (AMED) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e38 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shunsuke Goto More articles by this author Koji Tamada More articles by this author Masatoshi Eto More articles by this author Expand All Advertisement PDF downloadLoading ...

Genetics and Therapeutic Responses to Tumor-infiltrating Lymphocyte Therapy of Pancreatic Cancer Patient-derived Xenograft Models

Genetics and Therapeutic Responses to Tumor-infiltrating Lymphocyte Therapy of Pancreatic Cancer Patient-derived Xenograft Models

Anticancer effect of locally applicable aptamer-conjugated gemcitabine-loaded atelocollagen patch in pancreatic cancer patient-derived xenograft models.

We investigated the anticancer effect of the aptamer‐conjugated gemcitabine‐loaded atelocollagen patch in a pancreatic cancer patient–derived xenograft (PDX) model to propose a future potential adjuvant surgical strategy during curative pancreatic resection for pancreatic cancer. A pancreatic cancer PDX model was established. Animals were grouped randomly into a no‐treatment control group; treatment group treated with intraperitoneal gemcitabine injection (IP‐GEM) or aptamer‐conjugated gemcitabine (APT:GEM); and transplant with three kinds of patches: atelocollagen‐aptamer‐gemcitabine (patch I), atelocollagen‐inactive aptamer‐gemcitabine (patch II), and atelocollagen‐gemcitabine (patch III). Tumor volumes and response were evaluated based on histological analysis by H&E staining and Immunohistochemistry (IHC) was performed. Anticancer therapy–related toxicity was evaluated by hematologic findings. The patch I group showed the most significant reduction of tumor growth rate, compared with the no‐treatment group (p < 0.05). However, other treatment groups were not found to show significant reduction in tumor growth rate (0.05 < p < 0.1). There was no microscopic evidence suggesting potential toxicity, such as inflammation, nor necrotic changes in liver, lung, kidney, and spleen tissue. In addition, no leukopenia, anemia, or neutropenia was observed in the patch I group. This implantable aptamer‐drug conjugate system is thought to be a new surgical strategy to augment the oncologic significance of margin‐negative resection in treating pancreatic cancer in near future.

Anticancer Effect of Locally Applicable Aptamer-Conjugated Gemcitabine-Loaded Atelocollagen Patch in Pancreatic Cancer Patient-Derived Xenograft Models

Introduction: We investigated the anticancer effect of the aptamer-conjugated gemcitabine loaded atelocollagen patch in pancreatic cancer patient-derived xenograft model to propose a future potential adjuvant surgical strategy during curative pancreatic resection for pancreatic cancer. Method: Pancreatic cancer PDX model was established. Animals were grouped randomly (4∼9 mice per group, with 2 patient tumors) into no treatment control group, treated group with intraperitoneal Gemcitabine injection (IP-GEM), Aptamer-conjugated-Gemcitabine (APT:GEM) or transplant with 3 kinds of Patchs: Atelocollagen-Aptamer-Gemcitibin (Patch I), Atelocollagen-inactive Aptamer-Gemcitibine (Patch II), and Atelocollagen-Gemcitabine (Patch III). Tumor response was evaluated based on histological analysis by H&E staining and IHC was performed. Anticancer therapy-related toxicity was evaluated by hematologic finding. Results: Patch I group (necleolin-aptamer conjugated gemcitabine loaded atelocollagen-patch) showed the most significant reduction of tumor growth rate, comparing with no treatment group (p<0.05). However, other treatment groups were not found to show significant reduction in tumor growth rate (0.05< p <0.1). Patch I group also showed significant apoptotic process, but TUNNEL positive cancer cells were found along the superficial layer of the tumor surface, where therapeutic patch had been applied. There were no microscopic evidences suggesting potential toxicity, such as inflammation nor necrotic changes, in liver, lung, kidney, and spleen tissue. In addition, no leukopenia, no anemia, and no neutropenia were observed in Patch I group. Conclusions: This new implantable aptamer-drug conjugate system is thought to be new surgical strategy to augment the oncologic significance of margin-negative resection in treating pancreatic cancer in near future.

Overcoming Gemcitabine Resistance in Pancreatic Cancer Using the BCL-XL-Specific Degrader DT2216.

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.

Abstract 935: Preclinical activity of seribantumab in gastrointestinal cancers with NRG1 fusions

Abstract Background. Oncogenic rearrangements of the neuregulin 1 gene (NRG1) consist of a 5' partner fused to a 3' NRG1 sequence that retains the EGF-like domain, and are found in 0.2% of solid tumors including lung, breast and gastrointestinal (GI) cancers. Carcinomas of GI origin, including pancreatic and cholangiocarcinoma, represent around 20% of solid tumors harboring NRG1 fusions and there is no approved therapy for this group of cancers. The chimeric NRG1 oncoproteins bind to HER3/ERBB3 leading to trans-activation of other ERBB family members and trigger a signaling cascade that culminates in oncogenesis. Although targeting HER3 represents a rational therapeutic strategy for cancers harboring NRG1 fusions, this has remained relatively unexplored for NRG1 fusion-positive GI malignancies. In this study we investigated the efficacy of the anti-HER3 monoclonal antibody seribantumab in preclinical models of NRG1-driven GI cancers. Methods. We developed models of isogenic pancreatic cancer cells with NRG1 fusions by lentiviral-mediated cDNA expression of ATP1B1-NRG1 and SLC3A2-NRG1 fusions in immortalized pancreatic ductal cells (H6c7). Seribantumab efficacy was evaluated in isogenic cell lines and in patient-derived xenograft (PDX) models of pancreatic adenocarcinoma (CTG-0943, APP-NRG1 fusion) and intrahepatic cholangiocarcinoma (CH-07-0068, RBPMS-NRG1 fusion). Western blotting analysis was used to evaluate protein phosphorylation. Expression of NRG1 fusions was confirmed by RT-PCR and NGS. Results. Expression of NRG1 fusions in H6c7 cells resulted in enhanced phosphorylation of HER3 and AKT and increased sensitivity to afatinib, as compared to empty vector control cells (H6c7-EV). Treatment of H6c7-SLC3A2-NRG1 cells with seribantumab resulted in a dose-dependent inhibition of HER3 and AKT phosphorylation. Seribantumab treatment of H6c7-ATP1B1-NRG1 and H6c7-SLC3A2-NRG1 cells resulted in dose-dependent inhibition of cell growth with IC50 values of 0.05 and 0.2 µM, respectively. In contrast, growth of H6c7-EV cells was much less sensitive to seribantumab (IC50 &amp;gt; 1µM). Tumor growth inhibition was observed after administration of seribantumab to PDX mouse models of pancreatic adenocarcinoma and intrahepatic cholangiocarcinoma. While seribantumab (5 mg and 10 mg per dose, BIW) was equally effective to the clinical equivalent dose of afatinib (5 mg/kg QD) in the cholangiocarcinoma PDX model, the two doses of seribantumab were more effective than afatinib in the pancreatic cancer PDX model, causing tumor shrinkage of up to 55% (23-77% range). There was no shrinkage of afatinib-treated pancreatic PDX tumors. Our results here suggest that seribantumab is effective at reducing tumor growth in preclinical models of gastrointestinal cancers with NRG1 fusions. These data support the use of seribantumab to treat GI and other cancers with NRG1 fusions in the ongoing phase 2 CRESTONE study (NCT#04383210). Citation Format: Igor Odintsov, Allan J. Lui, Paul R. Bloom, Morana Vojnic, Shawn Leland, Marc Ladanyi, Romel Somwar. Preclinical activity of seribantumab in gastrointestinal cancers with NRG1 fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 935.

Anticancer effect of nucleoline-aptamer-conjugated gemcitabine loaded atellocollagen (IO401) in pancreatic cancer patient-derived orthotropic xenograft model

We investigated the anticancer effect and systemic effect of the atelocollagen (AC) patch coated nucleoline-aptamer-conjugated Gemcitabine (IO401 patch) by directly implanting to the tumor cell in pancreatic cancer patient-derived xenograft (PDX) model to purpose a future potential adjuvant surgical strategy during curative pancreatic resection for pancreatic cancer. Pancreatic cancer PDX model was established. Animals were grouped randomly (7 mice per group) into three types of patch transplantation groups: G1 = Null AC patch, G2 = Gemcitabine AC patch, G3 = IO401 patch. Tumor volume (length × width2, mm3), Tumor weight (mg), and Tumor inhibition rate [1-(Ti-To)/(average tumor volume of group) × 100, Ti = endpoint tumor volume, To = start tumor volume] were calculated. Anticancer therapy-related toxicity was evaluated by hematologic and histological findings. G3 (IO401 patch) showed the most significant reduction of tumor growth and tumor weight comparing with G1 (Null AC patch) and G2 (Gemcitabine AC patch) (p = 0.014, p = 0.018). G3 also showed the most significant tumor inhibition rate comparing with G1 and G2 (p = 0.011). G2 and G3 has the low necrosis proportion in histological finding comparing with G1 (p = 0.005, p < 0.05). Moreover, no leukopenia, no anemia, and no neutropenia were observed in G3. We demonstrated the anticancer effect of the IO401 patch by directly implanting to tumor cell in pancreatic cancer PDX model. This directaly implantable aptaber-drug conjugate system on tumor cell is expected to be a new surgical strategy to further increase the oncological importance of margin negative resection in pancreatic cancer surgery. Further research will be needed.

Identification, Structure-Activity Relationships of Marine-Derived Indolocarbazoles, and a Dual PKCθ/δ Inhibitor with Potent Antipancreatic Cancer Efficacy.

Protein kinases C (PKCs) are a family of serine/threonine kinases involved in various cellular processes, including proliferation, differentiation, cell survival, and apoptosis. Here, we report the identification, structure-activity relationship (SAR), and 3D-QSAR studies of 69 natural indolocarbazoles, including 15 new compounds, from marine streptomyces strains. Interestingly, we found that the chair conformational isomer of 7-oxo-staurosporine (compound 15) inhibited PKCθ more potently than the corresponding boat isomer. An evaluation of kinase selectivity and antitumor efficacy revealed that 15 was a potent dual PKCθ/δ inhibitor and that it could efficiently inhibit tumor growth in pancreatic cancer (PC) by inducing cellular apoptosis and suppressing the NF-κB/p-P65 pathway. In addition, we demonstrated that overexpression of p-PKCδ and p-P65 was associated with poor survival rates in patients with PC, and p-PKCθ expression also showed significant positive correlations with p-PKCδ and p-P65 levels. Finally, the PC patient-derived xenograft model further confirmed the potential anti-PC efficacy of 15.

Comparative study on contrast enhancement of Magnevist and Magnevist-loaded nanoparticles in pancreatic cancer PDX model monitored by MRI

BackgroundThe aim of this study was to compare contrast enhancement of Magnevist® (gadopentate dimeglumine (Mag)) to that of PEGylated Magnevist®-loaded liposomal nanoparticles (Mag-Lnps) in pancreatic cancer patient-derived xenograft (PDX) mouse model via magnetic resonance imaging (MRI).MethodsMag-Lnps formulated by thin-film hydration and extrusion was characterized for the particle size and zeta potential. A 21.1 T vertical magnet was used for all MRI. The magnet was equipped with a Bruker Advance console and ParaVision 6.1 acquisitions software. Mag-Lnps phantoms were prepared and imaged with a 10-mm birdcage coil. For in vivo imaging, animals were sedated and injected with a single dose (4 mg/kg) of Mag or Mag-Lnps with Mag equivalent dose. Using a 33-mm inner diameter birdcage coil, T1 maps were acquired, and signal to noise ratio (SNR) measured for 2 h.ResultsMag-Lnps phantoms showed a remarkable augmentation in contrast with Mag increment. However, in in vivo imaging, no significant difference in contrast was observed between Mag and MRI. While Mag-Lnps was observed to have fairly high tumor/muscle (T/M) ratio in the first 30 min, free Mag exhibited higher T/M ratio over the time-period between 30 and 120 min. Overall, there was no statistically significant difference between Mag and Mag-Lnp in rating MR image quality. Low payload of Mag entrapment by Lnps and restricted access of water (protons) to Mag-Lnps may have affected the performance of Mag-Lnps as an effective contrast agent.ConclusionThis study showed no significance difference in MRI contrast between Mag and Mag-Lnp pancreatic cancer PDX mouse models.

Nicotine Induces IL-8 Secretion from Pancreatic Cancer Stroma and Worsens Cancer-Induced Cachexia.

Smoking is highly associated with pancreatic cancer. Nicotine, the addictive component of tobacco, is involved in pancreatic cancer tumorigenesis, metastasis, and chemoresistance. This work aimed to describe the role of nicotine within the pancreatic cancer tumor microenvironment. Nicotine treatment was used in vitro to assess its effect on tumor-associated stromal cells and pancreatic cancer cells. Nicotine treatment was then used in a pancreatic cancer patient-derived xenograft model to study the effects in vivo. Nicotine induced secretion of interleukin 8 (IL-8) by tumor-associated stroma cells in an extracellular signal-regulated kinase (ERK)-dependent fashion. The secreted IL-8 and nicotine acted on the pancreatic cancer cell, resulting in upregulation of IL-8 receptor. Nicotine treatment of mice bearing pancreatic cancer patient-derived xenografts had significantly increased tumor mass, increased tumor-free weight loss, and decreased muscle mass. These represent important pathways through which nicotine acts within the tumor microenvironment and worsens pancreatic cancer-induced cachexia, potentially representing future therapeutic targets.

Abstract 3890: Pharmacodynamic and proteomic analysis of combined gemcitabine/nab-paclitaxel in patient-derived pancreatic cancer xenograft models

Abstract Gemcitabine (GEM) combined with albumin-bound paclitaxel nanoparticles (nab-PTX) is the first-line therapy for patients with metastatic pancreatic adenocarcinoma (PDAC). However, the in vivo mechanisms of drug action and interaction have not been investigated quantitatively for this combination. We evaluated the effects of GEM (100 mg/kg iv weekly) and nab-PTX (equivalent to 30 mg/kg PTX iv weekly), alone or combined, on tumor volume progression in 3 patient-derived xenograft (PDX) PDAC models in SCID mice. The MS1-based quantitative proteomic analysis (IonStar), which offers highly reproducible and accurate quantification with extremely low missing data (&amp;lt;0.5%), was conducted on Days 1, 4, and 8 after dosing to evaluate tumor pharmacodynamic responses in protein expression. Proteins were quantified based on species-specific peptides in order to identify responses of the PDAC cells (human) and tumor-associated stromal cells (murine). A population pharmacokinetic/pharmacodynamic model was developed to quantify tumor growth inhibition by the individual and combined drugs. Tumor growth kinetics were described by a composite function of exponential growth transitioning to linear growth. Treatment effects were represented by a 2nd-order, concentration-dependent parameter k that leads to cell death following delays arising from signal transduction. All 3 PDX models responded to the combination therapy, but with different sensitivities. PDX#14312 showed the least inhibition by the drugs alone (k[GEM]=13.6, k[PTX]=37.0 /day/mM), compared to the other PDXs (#18254: k[GEM]=68.1, k[PTX]=125; 18269: k[GEM]=112, k[PTX]=51.2 /day/mM). Compared to the more potent single-drug treatment for each PDX, the combination resulted in 1.33-fold greater cell death in #14312, 1.39 fold in #18269 and 1.19 fold in #18254. Model simulations showed distinct temporal profiles of cytotoxic signals in the 3 PDXs. Pathway analysis showed that the top changes mediated by the drug combination in PDX#14312 PDAC cells included extracellular matrix organization (FDR-corrected p-value = 7E-5), synthesis of nucleotide di- and triphosphates (p = 0.012), and interferon signaling (p = 0.04), whereas top changes in stroma included increased platelet activation (p = 3.8E-4) and extracellular matrix organization (p = 0.046). These processes represent potential contributors to the resistance of PDX#14312 to the drug combination. Top changes in #18269 and #18254, which were sensitive to the combination, included increased glycolysis (p = 0.038), decreased fatty acid beta-oxidation (p = 3E-5) and DNA replication (p = 0.0058) in PDAC cells, and increased complement activation (p &amp;lt; 1E-5) in stroma. Overall, inter-patient variability in PDAC responsiveness to GEM/nab-PTX therapy may be associated with tumor stress responses partially resembling wound healing processes. Citation Format: Jin Niu, Xue Wang, Shichen Shen, Ju Qu, Donald Mager, Robert M. Straubinger. Pharmacodynamic and proteomic analysis of combined gemcitabine/nab-paclitaxel in patient-derived pancreatic cancer xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3890.

Abstract 3621: Targeted hyaluronic acid nanoparticles improve treatment response in pancreatic cancer

Abstract Pancreatic cancer has poor response to chemotherapy with desmoplastic stroma identified as a delivery barrier. We have developed a nanoparticle (NP) drug system using an engineered ligand of receptor binding region of urokinase plasminogen activator (ATF) and the catalytic domain of metalloprotease (MMP14) carrying SN38 (CPT-11 analog). Hyaluronic acid (HA), a naturally occurring protein was chosen as the backbone to produce a biocompatible NP. HA can also bind to CD44, highly expressed in epithelial cancers. Dual uPAR, CD44 targeting allows for targeted delivery and facilitates receptor mediated endocytosis of NP-drug complex. MMP14 activity degrades extracellular matrix to deplete the stromal barrier. SN38 was encapsulated and recombinant ATFmmp14 conjugated to surface of self-assembled hyaluronic acid spheres (200nm) to form the complete particle (HANP). Cytotoxicity assays were conducted to determine IC50 in cell lines. Patient derived xenograft (PDX) model of a drug resistant pancreatic cancer was used for efficacy studies. Orthotopically implanted tumors were treated with HANP (10mg/kg SN38 dosage) weekly for 6 weeks via tail vein and overall survival compared to conventional therapy. HANP in-vitro cytotoxicity was greater than conventional Irinotecan (&amp;gt;80x) and liposomal irinotecan (&amp;gt;900x) in a PDX derived cell line (Table 1). In-vivo efficacy study demonstrated a significant improvement in survival of PDX bearing mice with HANP (n=9) (median survival 50 days), compared to FOLFIRINOX (n=9) and Gemcitabine-nab-Paclitaxel (n=9) (median survival 37 days each) and no treatment (n=13) (median survival 22 days). Combination of HANP with a modified-FOLFIRINOX regimen (n=9) led to median survival &amp;gt;72 days (p&amp;lt;0.001). HANP alone or in combination with a modified FOLFIRINOX led to significantly improved survival in a pancreatic cancer PDX model. Further studies are underway to evaluate preclinical PD/PK for eventual translation as targeted therapy for pancreatic cancer. In-vitro cytoxicity assay for IC50 determinationDrugIC50 (uM)HANP0.01SN-380.012Irinotecan0.8Liposomal Irinotecan9 Citation Format: Mohammad Raheel Jajja, Lei Zhu, Dazhi Wang, Charles A. Staley, Bassel El-Rayes, David A. Kooby, Lily Yang. Targeted hyaluronic acid nanoparticles improve treatment response in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3621.

Abstract 2792A: Preclinical activity of ADCT-601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting AXL-expressing tumors

Abstract AXL, a member of the tyrosine kinase receptor family TAM, is a transmembrane receptor overexpressed in many solid (e.g. lung, breast, pancreas, glioma and esophageal) and hematological malignancies (acute myeloid and chronic lymphocytic leukemia) and its overexpression is maintained in both primary tumors and metastasis. Moreover, expression and activation of AXL is associated with poor clinical prognosis and several studies suggest that overexpression of AXL results in resistance to both conventional chemotherapy and targeted therapies. All these features make AXL an attractive target for the development of an ADC to treat AXL-expressing cancers. ADCT-601 is an ADC composed of a humanized IgG1 antibody against human AXL, site-specifically conjugated using GlycoconnectTM technology to PL1601, which contains a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-601 in human cancer cell lines and xenograft models and to determine its safety and tolerability in the rat. In vitro, ADCT-601 demonstrated potent cytotoxicity in a panel of cell lines of different origin and levels of AXL, while its potency was strongly reduced in AXL-negative cell lines. In vivo, ADCT-601 showed potent anti-tumor activity in the human triple-negative breast cancer-derived MDA-MB-231 xenograft, expressing moderate levels of AXL (≈36,000 copies/cell). In this model, a single dose of ADCT-601 at 1 mg/kg resulted in strong and sustained anti-tumor activity and resulted in 5/10 partial responders (PR) and 4/10 tumor-free survivors (TFS) at the end of the study on day 60. In the SN12C model, a human renal cell carcinoma-derived xenograft with high level of AXL expression (≈88,000 copies/cell), single doses of ADCT-601 at 0.3, 0.6 or 1 mg/kg showed dose-dependent anti-tumor activity compared to the vehicle- and isotype control ADC-treated mice. At the highest dose, ADCT-601 resulted in 7/8 and 6/8 complete responder (CR) and TFS, respectively, at the end of the study on day 60. Moreover, ADCT-601 showed potent and durable anti-tumor activity in a pancreatic cancer patient-derived xenograft model expressing heterogeneous levels of AXL where each single dose of ADCT-601 tested (0.3, 0.6 and 1 mg/kg) resulted in complete eradication of the tumors at the end of the study on day 43. Conversely, none of the mice in the vehicle and isotype control ADC groups had any PR, CR or TFS. ADCT-601 was stable, well tolerated and showed a favorable PK profile in the rat with a half-life of 9 days and a MTD of 6 mg/kg. In conclusion, ADCT-601 demonstrated potent and specific in vitro and in vivo anti-tumor activity in various cancer-derived models with different levels of membranous AXL. ADCT-601 was stable and well tolerated in the rat, warranting further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Simon Chivers, Paul W. Hogg, Charlie Britten, Sandamali Dissanayake, Peter Tyrer, Francois Bertelli, Ian Hutchinson, Luke Masterson, Phil Howard, John A. Hartley, Patrick H. van Berkel. Preclinical activity of ADCT-601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) targeting AXL-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2792A.

Individualized drug screening based on next generation sequencing and patient derived xenograft model for pancreatic cancer with bone metastasis.

The efficacy of traditional chemoradiotherapies for pancreatic cancer remains limited, and no effective targeted therapies or screening tests are currently available. Therefore more individualized drug screening is warranted for the clinical treatment of pancreatic cancer. A patient-derived xenograft (PDX) model of pancreatic cancer bone metastasis was established, and next-generation sequencing (NGS) was used to investigate the molecular characteristics of the cancer and screen for potential drugs. Immunohistochemical analysis was performed to validate that the PDX retained the molecular characteristics from the patient. Using NGS technology, 13 pancreatic-cancer-associated polymorphisms/mutations were identified out of 416 genes sequenced. Based on the sequencing results and associated literatures, AZD6244, a highly selective inhibitor against mitogen-activated protein kinase kinase 1 (MEK1), was chosen as a potential therapy. AZD6244, a highly selective MEK1 inhibitor, was evaluated as effective for the pancreatic cancer PDX model, and thus may provide potential efficacy in the clinical treatment of the patient with pancreatic cancer investigated in the present study. The feasibility of the novel NGS-PDX based drug-screening pattern was demonstrated, and has a potential to improve individualized treatment for cancer.

Abstract 4937: Comparison of orthotopic and subcutaneous implantation of pancreatic cancer patient-derived xenograft models for drug development

Abstract Patient derived xenograft (PDX) models of pancreatic cancer provide an excellent platform for understanding human disease, characterizing tumor-initiating cells, and testing of novel therapeutics. Herein, we compared the natural pancreatic microenvironment via orthotopic implantation and the traditional approach of subcutaneous implantation for effects on PDX tumor growth, tumor morphology, cell population diversity, tumorigenic potential, metastatic potential, and response to therapeutics. Two approaches were used: (1) primary human cancer tissue fragments were implanted in orthotopic and subcutaneous environments; and, (2) PDX previously established by subcutaneous engraftment were reimplanted into either an orthotopic or a subcutaneous environment. We report that orthotopically-engrafted primary PDX more closely reflect the gross tumor morphology of the original patient than subcutaneously-derived tumors. In models derived from subcutaneous engraftment, variation of tumor morphology, tumor growth, cell population diversity, and tumorigenic potential between tumors grown in orthotopic and subcutaneous environments were dependent on PDX line; and, stromal infiltration and mucin production were more prominent in the orthotopic background for some PDX models. In addition, we observe increased metastatic potential in orthotopic tumor-bearing mice compared with their subcutaneous counterparts. Tumor metastasis occurred in distal filtering organs, including the lung, liver, lymph nodes, and peritoneal cavity, among others. This result is consistent with the observation of increased circulating tumor cells in the blood of orthotopic tumor bearing mice. Comparison of therapeutic efficacy with gemcitabine chemotherapy and antibody-drug conjugate (ADC) therapy in orthotopic and subcutaneous tumors showed therapeutic response was dependent on PDX line. Interestingly, for PDX lines that exhibited morphological differences between orthotopic and subcutaneous backgrounds, treatment was less effective in the orthotopic microenvironment suggesting the microenvironment alters the ability of therapeutics to impede tumor growth. Reduced therapeutic response was also found to be consistent with reduced ADC uptake and vascularity in orthotopic tumors. Taken together, the orthotopic PDX model serves as a more accurate representation of human pancreatic cancer by displaying stromal-rich morphology, exhibiting metastatic cell behavior, and recapitulating therapeutic response challenges observed in patients. Citation Format: Sarah L. Fong, Marybeth A. Pysz, Kristen D. McKnight, Nicole Taylor, Regina Nieu, Juliet Markeson, Hanna Rammoth, Archana Dilip, Kayla Fasano, Emily Janke, Erica Anderson, Holger Karsunky, Scott Dylla. Comparison of orthotopic and subcutaneous implantation of pancreatic cancer patient-derived xenograft models for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2017-4937

Abstract 4071: FGFR1 inhibitor reverses Smoothened inhibition-induced epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma

Abstract The majority of Pancreatic cancer (PaCA) patients present with metastatic disease and receive little benefit from chemotherapy. Although nanoparticulate carriers can potentially deposit sustained and high doses of chemotherapeutics in tumors, PaCA tumors are poorly perfused, which limits drug deposition. Ten days of oral dosing with NVP-LDE225 (40 mg/kg), an inhibitor of the sonic hedgehog (SHH) pathway (sHHI), increased tumor deposition of blood-borne, fluorescently labeled, sterically-stabilized lipidic (SSL) nanoparticles by enhancing the perfusion and permeability of the otherwise-impermeable microvasculature of PaCA patient-derived xenograft (PDX) tumors. However, literature indicates sHH ablation is associated with increased Epithelial-to-Mesenchymal (EMT) transition and invasiveness in PaCA. Here we investigated the compensatory role of FGFR1 in sHHI-induced tumor progression. Four to 8 daily treatments with the sHHI resulted in no significant reduction of tumor volume, but markedly increased the ratio of tumor cells (human mitochondria+) to Collagen1 in the stroma, in two s.c. low-passage PDX models of varying stromal content. Immunostaining of sHHI-treated tumors revealed no alteration in total FGFR1 expression, but showed a significant elevation in the (1) percent of FGFR1+ pixels correlating with DAPI+ nuclei in tumor (human mitochondria+) cells and stromal cells, (2) number of vimentin+ (EMT) cells, and (3) proliferative index marked by Ki67+ nuclei, compared to controls, suggesting accelerated tumor progression. Nuclear FGFR1 was associated with vimentin+ cells, but was excluded from the nuclei of Ki67+ cells in all groups, implicating nuclear translocation of FGFR1 in EMT. NVP-BGJ398, a small-molecule pan-FGFR inhibitor (FGFRI), administered p.o. for 3 days (15 mg/kg/day) after 7d of sHHI-pretreatment (1) significantly reduced nuclear localization of FGFR1 compared to sHHI-treated animals, (2) reversed the ratio of human mitochondria+ tumor cells to Collagen 1+ stroma, (3) depleted vimentin+ structures by &amp;gt;2 fold, and (4) reduced Ki67+ cells to control levels. FGFRI without sHHI-pretreatment resulted in decreased nuclear FGFR1 relative to controls, but did not impact proliferation, EMT, or tumor cell-to-stroma ratio. Finally, the sHHI/FGFRI sequence increased functional (FITC-L. esculentum lectin+) tumor vessel density and area the perfused by vascular permeability probes (fluorescent SSL) compared to both controls and sHHI-treated animals. FGFRI administration in vehicle-treated controls had no impact on vascular permeability or perfusion. We conclude that the combination of sHHI in sequence with FGFRI may reverse potentially deleterious effects of sHHI and mediate a transient enhancement of tumor permeability, and increase deposition and penetration of nanoparticulate SSL drug formulations into pancreatic cancers. Citation Format: Tista Roy Chaudhuri, Qingxiang Lin, Ninfa L. Straubinger, Wen Wee Ma, Robert M. Straubinger. FGFR1 inhibitor reverses Smoothened inhibition-induced epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2017-4071

Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth.

Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo fatty acid synthesis, and its ACC1 isoform is overexpressed in pancreatic and various other cancers. The activity of many oncogenic signaling molecules, including WNT and Hedgehog (HH), is post-translationally modified by lipidation. Here, we report that inhibition of ACC by a small molecule inhibitor, BAY ACC002, blocked WNT3A lipidation, secretion, and signaling. In pancreatic cancer cells, where WNT and HH are key oncogenic drivers, ACC inhibition simultaneously suppressed WNT and HH signaling, and led to anti-proliferative effects. Treatment with ACC inhibitors blocked tumor growth and converted the poorly differentiated histological phenotype to epithelial phenotype in multiple cell line-based and patient-derived pancreatic cancer xenograft models. Together, our data highlight the potential utility of ACC inhibitors for pancreatic cancer treatment, and provide novel insight into the link between upregulated de novo fatty acid synthesis in cancer cells, protein lipidation, and oncogenic signaling.