Patient-derived Tumor Xenograft Mouse Models Research Articles

Epstein-Barr Virus-Encoded MicroRNA-BART18-3p Promotes Colorectal Cancer Progression by Targeting De Novo Lipogenesis.

The Epstein-Barr virus (EBV) genome encodes a cluster of 22 viral microRNAs, called miR-BamHI-A rightward transcripts (miR-BARTs), which are shown to promote the development of cancer. Here, this study reports that EBV-miR-BART18-3p is highly expressed in colorectal cancer (CRC) and is closely associated with the pathological and advanced clinical stages of CRC. Ectopic expression of EBV-miR-BART18-3p leads to increased migration and invasion capacities of CRC cells in vitro and causes tumor metastasis in vivo. Mechanistically, EBV-miR-BART18-3p activates the hypoxia inducible factor 1 subunit alpha/lactate dehydrogenase A axis by targeting Sirtuin, which promotes lactate accumulation and acetyl-CoA production in CRC cells under hypoxic condition. Increased acetyl-CoA utilization subsequently leads to histone acetylation of fatty acid synthase and fatty acid synthase-dependent fat synthesis, which in turn drives de novo lipogenesis. The oncogenic role of EBV-miR-BART18-3p is confirmed in the patient-derived tumor xenograft mouse model. Altogether, the findings define a novel mechanism of EBV-miR-BART18-3p in CRC development through the lipogenesis pathway and provide a potential clinical intervention target for CRC.

SiRNA-circFARSA-loaded porous silicon nanomaterials for pancreatic cancer treatment via inhibition of CircFARSA expression

Novel functions and involvement of circFARSA have not been reported in pancreatic cancer; in addition, its inhibitor screening has not yet been conducted. The purpose of this study was to (1) verify circFARSA as a novel anti-cancer target for pancreatic cancer and (2) to prepare a novel anti-pancreatic cancer agent targeting circFARSA. In this study, we designed and synthesized a small interfering RNA (siRNA, named siRNA-circFARSA), which specifically inhibits circFARSA expression. Using liposomes and porous silicon nanoparticles (pSiNPs) as siRNA delivery system, we prepared liposome-siRNA-circFARSA and pSiNP-PEI-siRNA-circFARSA and investigated their anti-cancer mechanism by quantitative real-time PCR and western blotting. Cell proliferation curves and transwell migration assays were performed to investigate the effect of siRNAs proliferation and migration capabilities of cancer cells. Patient-derived tumor xenograft mouse models were used to investigate the anti-cancer effects in vivo. The data showed that both liposome-siRNA-circFARSA and pSiNP-PEI-siRNA-circFARSA (Si: 0.7 µg/mL) significantly inhibited the proliferation and migration of pancreatic cancer cells in vitro. However, the biological safety and in vivo anti-cancer effects of pSiNP-PEI-siRNA-circFARSA (Si: 22.4 µg/mL) were higher than those of liposome-siRNA-circFARSA. The results showed that siRNA-circFARSA could inhibit the expression of circFARSA and then BCL-2 protein expression, thereby leading to pancreatic cancer cell apoptosis after transportation into pancreatic cancer cells. Therefore, this study provides tools for pancreatic cancer treatment in the future, as it (1) verified circFARSA as a novel target for pancreatic cancer treatment, and (2) prepared a novel anti-pancreatic cancer agent (pSiNP-PEI-siRNA-circFARSA).

Knockdown of eukaryotic translation initiation factor 5A2 enhances the therapeutic efficiency of doxorubicin in hepatocellular carcinoma cells by triggering lethal autophagy.

Hepatocellular carcinoma (HCC) is an invasive malignant neoplasm with a poor prognosis. The development of chemoresistance severely obstructs the chemotherapeutic efficiency of HCC treatment. Therefore, understanding the mechanisms of chemoresistance is important for improving the outcomes of patients with HCC. Eukaryotic translation initiation factor 5A2 (eIF5A2), which is considered to be an oncogene, has been reported to mediate chemoresistance in various types of cancer; however, its precise role in HCC remains unclear. Accumulating evidence has suggested that autophagy serves a dual role in cancer chemotherapy. The present study aimed to investigate the role of autophagy in eIF5A2-mediated doxorubicin resistance in HCC. High expression levels of eIF5A2 in human HCC tissues were observed by immunohistochemistry using a tissue microarray, which was consistent with the results of reverse transcription-quantitative PCR analysis in paired HCC and adjacent healthy tissues. HCC patient-derived tumor xenograft mouse model was used for the in vivo study, and knockdown of eIF5A2 effectively enhanced the efficacy of doxorubicin chemotherapy compared with that in the control group. Notably, eIF5A2 served as a repressor in regulating autophagy under chemotherapy. Silencing of eIF5A2 induced doxorubicin sensitivity in HCC cells by triggering lethal autophagy. In addition, 5-ethynyl-2′-deoxyuridine, lactate dehydrogenase release assay and calcein-AM/PI staining were used to determine the enhanced autophagic cell death induced by the silencing of eIF5A2 under doxorubicin treatment. Suppression of autophagy attenuated the sensitivity of HCC cells to doxorubicin induced by eIF5A2 silencing. The results also demonstrated that knockdown of the Beclin 1 gene, which is an autophagy regulator, reversed the enhanced autophagic cell death and doxorubicin sensitivity induced by eIF5A2 silencing. Taken together, these results suggested eIF5A2 may mediate the chemoresistance of HCC cells by suppressing autophagic cell death under chemotherapy through a Beclin 1-dependent pathway, and that eIF5A2 may be a novel potential therapeutic target for HCC treatment.

Abstract 1675: Enhanced antitumor effect of dual EGFR and VEGFR2 inhibition in EGFR-mutated non-small cell lung cancer (NSCLC) patient-derived tumor xenograft (PDX) models

Abstract Background: NSCLC accounts for nearly 85% of all lung cancers. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective for EGFR mutated NSCLCs, but these drugs often stop working after EGFR acquires additional mutations. Preclinical and clinical data suggest a stronger antitumor effect with dual blockade of the EGFR and vascular endothelial growth factor (VEGF) pathways in EGFR-mutated NSCLC. RELAY, a randomized, double-blind, phase 3 trial, demonstrated superior progression-free survival (PFS) with ramucirumab (RAM) + erlotinib compared with placebo + erlotinib in patients with untreated EGFR-mutated advanced NSCLC. Additionally, RAM was tested in a Phase 1 study with the third-generation irreversible EGFR TKI osimertinib (OSI); AstraZeneca) in NSCLC patients with advanced T790M-positive EGFR mutant tumors after progression on first-line EGFR TKI therapy. OSI can be used with L858R, exon 19 del and T790M EGFR mutant tumors. We investigated the combined use of OSI+RAM in EGFR mutated NSCLC PDX mouse models. Materials and Methods: The antimurine VEGFR2 antibody DC101 (40 mg/kg, BIW) and the EGFR TKI OSI (10 mg/kg, QD) were used in this study. Twenty-seven PDX NSCLC mouse models carrying EGFR activating and/or T790M resistance mutations, commonly detected in patients with advanced NSCLC, were evaluated at 3 independent sites. Mice were divided into 4 treatment arms: vehicle (PBS), DC101, OSI and combination (DC101+OSI). Each study arm included two transplanted mice per model. Sensitivity to DC101 and OSI monotherapies, enhancement of anti-tumor activity with the combination therapy and durability of the response were evaluated. Desirability score, a weighted composite score of six features of tumor growth curves including best median % response, number of days in best response category, time to doubling, re-growth rate (last 5 observations) relative to vehicle growth rate, last observed tumor volume and last observed day, was calculated for each treatment arm. Analysis of variance was used to compare summary scores per animal across treatments. Results: OSI alone and OSI+DC101 combination therapy had stronger antitumor effects than DC101 monotherapy in significantly more models 11 vs 1 and 12 vs 0, respectively. A small but consistent shift in favor of OSI+DC101 vs OSI was observed across most of the models. Paired t-test analyses showed a moderate but significant benefit with combination OSI+DC101 vs OSI. Overall desirability score was statistically improved with OSI+DC101 compared to either single agent. Conclusions: Dual inhibition of EGFR and VEGFR2 with OSI+DC101 had a stronger antitumor effect than those of the respective monotherapies in EGFR mutated NSCLCs in vivo. Future studies will investigate the molecular mechanisms underlying the enhanced antitumor effect of OSI+DC101 combination therapy. Citation Format: Sudhakar Chintharlapalli, Anthony Fischl, Chun Ping Yu, Philip Iversen. Enhanced antitumor effect of dual EGFR and VEGFR2 inhibition in EGFR-mutated non-small cell lung cancer (NSCLC) patient-derived tumor xenograft (PDX) models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1675.

Establishment of a Liver Transplant Patient-derived Tumor Xenograft (PDX) Model Using Cryopreserved Pancreatic Ductal Adenocarcinoma.

There is rapid progression and widespread use of patient-derived tumor xenografts (PDX) in translational pancreatic cancer research. This study aimed to establish a liver transplant PDX model using cryopreserved primary pancreatic ductal adenocarcinoma (PDAC). Primary PDAC from 10 patients were cryopreserved and transplanted into immunodeficient mice using the liver pocket method. H&E staining and immunohistochemical staining, such as Ki-67, p53, Smad4, and MUC1 were used to evaluate engraftment and histological similarities. Patient-derived xenograft placement was successful in six cases (60%), and 10 mice (33.3%). The Ki-67 index of primary PDAC and the cryopreservation duration were significantly related to successful engraftment (p=0.003 and p=0.007, respectively). In this study, we succeeded in establishing a liver transplant PDX mouse model as a preclinical platform. The successful engraftment was affected by the cryopreservation duration and could be detected by the Ki-67 index.

Establishment of patient-derived tumor xenograft (PDX) model as artificially-created liver metastasis using cryopreserved pancreatic ductal adenocarcinoma.

729 Background: Translational research using patient-derived tumor xenograft (PDX) model is progressing rapidly, and is also becoming widespread in pancreatic cancer research. The purpose of this study was to establish the liver transplant PDX model as artificially-created liver metastasis with cryopreserved primary pancreatic ductal adenocarcinoma(PDAC). Methods: The primary PDAC from 10 patients were cryopreserved and transplanted into NSG mice using liver pocket method. For engraftment and similarity evaluation, H&E staining and immunohistochemical staining such as Ki-67, p53, SMAD4, and MUC1 were performed. Results: Patient-derived xenograft was succeeded in 6 cases (60%), 10 mice (33.3%). Ki-67 index of primary PDAC and the interval of cryopreservation were significantly related to successful engraftment, respectively (p = 0.003, p = 0.007). Conclusions: In this study, we succeeded in establishing a liver transplant PDX mouse model as a preclinical platform. The factors such as Ki-67 index and the interval of cryopreservation would affect the successful establishment.

Primary Tumor Site Specificity is Preserved in Patient-Derived Tumor Xenograft Models.

Patient-derived tumor xenograft (PDX) mouse models are widely used for drug screening. The underlying assumption is that PDX tissue is very similar with the original patient tissue, and it has the same response to the drug treatment. To investigate whether the primary tumor site information is well preserved in PDX, we analyzed the gene expression profiles of PDX mouse models originated from different tissues, including breast, kidney, large intestine, lung, ovary, pancreas, skin, and soft tissues. The popular Monte Carlo feature selection method was employed to analyze the expression profile, yielding a feature list. From this list, incremental feature selection and support vector machine (SVM) were adopted to extract distinctively expressed genes in PDXs from different primary tumor sites and build an optimal SVM classifier. In addition, we also set up a group of quantitative rules to identify primary tumor sites. A total of 755 genes were extracted by the feature selection procedures, on which the SVM classifier can provide a high performance with MCC 0.986 on classifying primary tumor sites originated from different tissues. Furthermore, we obtained 16 classification rules, which gave a lower accuracy but clear classification procedures. Such results validated that the primary tumor site specificity was well preserved in PDX as the PDXs from different primary tumor sites were still very different and these PDX differences were similar with the differences observed in patients with tumor. For example, VIM and ABHD17C were highly expressed in the PDX from breast tissue and also highly expressed in breast cancer patients.

PS02.239: SPECIFIC INHIBITION OF BONE MORPHOGENIC PROTEIN (BMP4) AS A POTENTIAL THERAPEUTIC STRATEGY FOR ESOPHAGEAL ADENOCARCINOMA

Abstract Background In Western countries, the highly malignant Esophageal adenocarcinoma (EAC) have the most dramatically rising incidence of all malignancies. BMP4 is a growth factor important for carcinogenesis. We found that BMP4 is aberrantly expressed in Barret's esophagus, the pecursor lesion of EAC, and that together with CDX2 drives the intestinalization of epithelial metaplasia. However, its role in esophageal adenocarcinoma (EAC) remains uncertain. Methods Method: To elucidate whether BMP4 is involved in malignancy in EAC we used an RNA sequencing database of 56 EAC treatment naive endoscopic biopsies to investigate if there is a subgroup of cancers with high BMP signaling. We validated results by qPCR and immunohistochemistry in matching tumor samples. Next we used our recently developed effective and highly specific anti-BMP4 antibodies(1,2) to study the effect of inhibition of BMP4 on both in vitro as well as in vivo models of EAC. Results Using a gene set that was recently published for BMP signaling, we were able to distinguish a subgroup of EAC patients with increased BMP signaling. By IHC we confirmed that 70% of EAC tumors express BMP4 at the protein level. We found that patients with high levels of BMP4 expression tend to have a poorer recurrence-free survival compared to patients with low BMP4 expression, which suggests a more aggressive tumor behavior in BMP4 expressing EAC tumors. Most importantly, inhibition of BMP4 function in EAC cells by our recently developed anti-BMP4 antibodies lead to an increase in chemo-sensitivity and a decreased in invasive and migratory capabilities in vitro. Preclinical in vivo studies with a patient-derived tumor xenograft mouse model of an EAC tumor confirmed that anti-BMP4 antibodies can effectively reduce tumor growth and synergistically act with chemotherapy agents. Conclusion We identified a subgroup of EAC with increased BMP signaling. Our studies support a role of BMP4 in chemo-resistance and invasiveness in EAC, and indicate that inhibition of BMP4 with highly our specific llama-derived antibodies is an attractive therapy for improving outcomes of EAC. Disclosure All authors have declared no conflicts of interest.

Targeting Proteome-Scale Networks to Design and Synthesize Potent Anticancer and Cell-Specific Immunomodulatory Compounds

Currently, there are no high-throughput experiments to quantify all biochemical interactions of small molecules in an organism (human) that is essential to ascertain both toxicity and efficacy of a drug. Furthermore, the synthesis of compound/drug libraries explores diverse chemical space that is unrelated to the disease with no guidance from disease-specific biological pathways (targets and antitargets). Recognizing this unexplored area, we have developed proteome-scale target/antitarget guided lead optimization methods (CANDESIGN) for discovery and de novo design of safe chemical libraries. Selection of target/anti-target networks are identified iteratively using machine learning on experimental data for desired cellular phenotypes in vitro and in vivo. We model interactions with proteomes from different organisms (currently 48,278) to infer homology of compound/drug behavior. Our approach has been verified for drug repurposing, design then synthesis of potent non-toxic anticancer pathway-specific libraries, and compounds that alter specific cell functions in vivo. Repurposed leads are identified from ∼1 billion predicted compound-proteome interactions with ∼35% accuracy across nine indications (immunological, metabolic, infectious). Compound libraries synthesized for specific hormone signaling pathways result in picoMolar anticancer leads to combat resistant cancers. Our leads are non-toxic in vitro and in vivo and more efficacious than current treatments on patient-derived tumor xenograft mouse models. Similarly, we designed compounds to specifically change function of Myeloid Derived Suppressor Cells (MDSC) from upregulated gene networks of monocytic MDSCs in the cancer microenvironment. Cancer cell lines (human, mouse, dog) are insensitive to our MDSC-specific library in vitro but exerts antitumor impact in vivo via immunomodulation. We observed decreased frequencies of MDSC expressing suppressive functional markers (iNOS, PD-L1) and increased frequency of IFN-γ+CD8+ T-cells for antitumor activity. We conclude that proteome-scale networks result in safer “chemical probes” to effect similar pathways across different diseases therapeutically.

Abstract 623: Feasibility of surgical orthotopic implantation for establishment of patient-derived liver metastatic uveal melanoma xenograft model in NSG mouse

Abstract The prognosis of metastatic uveal melanoma (UM) is extremely poor. Approximately 90% of patients with metastatic UM die from hepatic metastasis. Controlling hepatic metastases has been a rational approach to extend survival. This raises an urgent need to develop a novel mouse model for further investigation. Patient-derived tumor xenograft (PDX) mouse model provides potential benefit through a personalized medicine approach. This model may be useful for predictions of clinical outcomes, drug efficacy and biomarker identification. Most PDX models, including UM PDX models, are made by ectopic subcutaneous implantation. Subcutaneous PDX models have some obstacles in translational research due to differences in anatomic microenvironment from tumor origin, low engraftment rate, and slow tumor growth. Our goal was to develop an orthotopic PDX model of liver metastatic UM which would overcome these difficulties. In developing our model, we first compared the differences in growth in a liver with a subcutaneous site using two UM cell lines (UM001 and UM004) in NSG mice, to ascertain which site is a more suitable tumor microenvironment. Second, we investigated the feasibility of surgical orthotopic implantation (SOI) of tumor masses to develop a practical liver tumor xenograft mouse model with UM cell lines in 20 mice. Finally, we tried to establish an orthotopic PDX mouse model of liver metastatic UM in 5 mice. We also evaluated concordance for tumor characteristics between pre-implant and post-implant tumors. Our results showed that the liver was a more suitable microenvironment to grow the two UM cell lines compared to the subcutaneous site. UM001 engrafted in the liver, whereas it did not engraft in the subcutaneous site. UM004 displayed more rapid growth in the liver compared to the subcutaneous site. A new SOI of tumor chunk was created to develop a practical liver tumor xenograft model. Donor tumors, which were generated in the liver after injecting UM cell lines, were surgically transplanted into the liver of recipient mice. The new SOI was successfully performed in all 20 mice without adverse events. Recipient mice formed tumors in the liver after SOI. For comparison, the tumor chunks of UM cell lines were also implanted subcutaneously, in which we were able to show that the growth was more rapid in the liver. Both donor and recipient tumors had matching tumor characteristics in histological and proteomic analysis. We established an orthotopic PDX mouse model of liver metastatic UM with a new SOI method. The success rate of engraftment was 80% (4/5) within 6 months. Tumor characteristics of both patient's original tumors and xenograft mouse tumors matched in histological, genomic and proteomic analysis. This new SOI method can establish a patient-derived liver metastatic uveal melanoma xenograft model in NSG mouse at a highly successful rate. Citation Format: Ken Kageyama, Masahiro Ohara, Kengo Saito, Shinji Ozaki, Mizue Terai, Andrew E. Aplin, Michael J. Mastrangelo, Takami Sato. Feasibility of surgical orthotopic implantation for establishment of patient-derived liver metastatic uveal melanoma xenograft model in NSG mouse. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 623.

MET overexpression and amplification define a distinct molecular subgroup for targeted therapies in gastric cancer.

Currently, only trastuzumab, ramucirumab, and apatinib effectively treat gastric cancer. Thus, additional novel targets are required for this disease. We investigated the immunohistochemical and fluorescence in situ hybridization expression of MET, ROS1, and ALK in four gastric cell lines and a cohort of 98 gastric cancer patients. Crizotinib response was studied in vitro and in vivo. Crizotinib potently inhibited in vitro cell growth in only one cell line, which also showed MET amplification. A positive correlation between crizotinib sensitivity and MET overexpression was observed (P=0.045) in the histoculture drug response assay. Meanwhile, patient-derived tumor xenograft mouse models transplanted with tissues with higher MET protein expression displayed a highly selective sensitivity to crizotinib. In the 98 patients, MET overexpression was found in 42 (42.9%) and MET was amplified in 4 (4.1%). ROS1 and ALK overexpression were found in 25 (25.5%) and 0 patients, respectively. However, none of the patients screened harbored ALK or ROS1 rearrangements. No significant association was found between overall survival and MET or ROS1 status. We also observed a stage IV gastric cancer patient with MET amplification who experienced tumor shrinkage and clinical benefit after 3weeks of crizotinib as fourth-line treatment. Crizotinib may induce clinically relevant anticancer effects in MET-overexpressed or MET-amplified gastric cancer patients.