Orthotopic Implantation Model Research Articles

A Clinically Applicable Gene-Expression Classifier Reveals Intrinsic and Extrinsic Contributions to Consensus Molecular Subtypes in Primary and Metastatic Colon Cancer.

Four consensus molecular subtypes (CMS1-4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer-related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC). We developed a colorectal cancer-focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer in vivo at relevant sites. Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer. We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.

Abstract 5082: SIRT1 functions as a double-edged sword in prostate cancer

Abstract SIRT1 is a NAD+ dependent deacetylase known to regulate a plethora of biological processes through posttranslational regulation of proteins including those that function as tumor promoters and suppressors. In order to define the role of SIRT1 in prostate pathogenesis, we used 2 mouse models: (i) PTEN knockout (PTENKO) mouse model by pharmacological activation of SIRT1 with resveratrol (RES) a known activator of SIRT1, & (ii) orthotopic implantation model with genetic silencing of SIRT1 (SIRT1 shRNA). We also used genetic and pharmacologic inhibition of SIRT1 in cell culture models to understand the mechanism. We tested whether SIRT1 modulation is beneficial when targeted early (before the establishment of prostatic lesions) or late (after the establishment of prostatic lesions) in the PTENKO model. RES intervention was initiated in 4-5-week (early intervention) and 10-15-week-old (late intervention) PTENKO mice. Analyses of samples collected longitudinally during progression revealed that early intervention with RES reduced incidence of high-grade prostate intraepithelial neoplastic lesions (HGPIN) when given for 14 weeks with no significant difference at 7 or at 11 weeks. On the other hand, late intervention after the establishment of HGPIN lesions with RES had no beneficial effect. Importantly, longer treatment duration (28 weeks) resulted in significantly increased incidence of invasive prostate carcinoma. Furthermore, RES had no significant effect on the development of orthotopic prostate tumors following implantation of LNCaP cells in nude mice. In contrast, orthotopic implantation of SIRT1 stably silenced LNCaP cells showed significant impairment in tumor development. Immunohistochemical evaluation showed nuclear localization of SIRT1 in human prostate tumors and was associated with increased risk of biochemical recurrence. Mechanistic investigations revealed (i) suppression of AR signaling in hormone-sensitive LNCaP but not in castration-resistant 22Rv1 cells; (ii) RNAseq coupled with gene ontology enrichment analysis using SIRT1 silenced cells under conditions of androgen stimulation and inhibition identified genes involved in cell cycle checkpoint and senescence as top pathways affected by SIRT1 loss of function. In silicoanalysis shows that the identified SIRT1-regulated targets are associated with disease aggressiveness and poor disease-free survival. Taken together these data demonstrate that (i) SIRT1 plays a contextual role during prostate pathogenesis by functioning as tumor suppressor during early stage and as a tumor promoter during late stage; (ii) SIRT1 inhibition suppresses tumor development and (iii) RES is a better preventive than therapeutic agent. Therefore, our findings offer promising avenues to develop SIRT1-regulated pathways as novel therapeutic targets to inhibit prostate cancer recurrence. Supported by CPRIT Training Grant RP 170345 (SH) and CPRIT RP 150166 (APK) Citation Format: Shih-Bo Huang, Dinesh Thapa, Roble G. Bedolla, Amanda R. Muñoz, Xiaoyu Yang, Paul Rivas, Robert L. Reddick, Hiroshi Miyamoto, Rita Ghosh, Addanki Pratap Kumar. SIRT1 functions as a double-edged sword in prostate 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 5082.

Abstract 522: PI3K/Akt signaling in tumor cells promotes immune evasion by limiting infiltration, recognition and killing by T cells

Abstract The presence of tumor-infiltrating T cells is associated with favorable patient outcomes, yet most pancreatic cancers are immunologically silent and resistant to currently available immunotherapy. In this study, a syngeneic orthotopic implantation model of pancreatic cancer was used to show that PI3K/Akt signaling in tumor cells down-regulate immunogenicity to evade T cell surveillance. Genetic silencing of PI3K catalytic isoform Pik3ca in KrasG12D/Trp53R172H-driven pancreatic tumors leads to infiltration of T cells, complete tumor regression, and 100% survival of immunocompetent host mice. By contrast, Pik3ca-null tumors implanted in T cell-deficient mice progress and kill 100% of the animals. Adoptive transfer of tumor antigen-experienced T cells eliminates Pik3ca-null tumors in immunodeficient mice. Pik3ca silencing or inhibition of its effector, Akt, increases the expression of MHC Class I and CD80 on the surface of murine and human pancreatic cancer cells. Down-regulating MHC Class I and CD80 expression, or increasing Akt activity, in Pik3ca-null tumors increases their susceptibility to T cell-mediated regression in vivo. These results indicate that PI3K/Akt signaling in tumor cells limits T cell recognition and clearance of pancreatic cancer. Strategies that selectively target the PI3K/Akt signaling pathway in tumor cells may improve immunotherapy for pancreatic cancer. Citation Format: Nithya Sivaram, Patrick McLaughlin, Ya-Ping Jiang, Lisa Ballou, Kien Pham, Chen Liu, Adrianus van der Velden, Richard Lin. PI3K/Akt signaling in tumor cells promotes immune evasion by limiting infiltration, recognition and killing by T cells [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 522.

Antisense oligonucleotide against insulin-like growth factor I receptor inhibits tumor growth in human hepatocellular carcinoma orthotopic implantation models and its influence on the blood indicators in nude mice in vivo

Objective To investigate the inhibitory effect of antisense oligonucleotide against insulin-like growth factor 1 receptor (IGR-1R) on tumor growth in human hepatocellular carcinoma orthotopic implantation models and its influence on the blood indicators in nude mice. Methods We did the experiment 3 times, and 40 models for each time were divided into 5 groups randomly: NS control group, three IGF-1R antisense oligdeoxynucleotides (ASODN) [75, 50, 25 mg/(kg·day)] groups and 5-fluorouracil (5-Fu) control group. Eighty nude mice served as normal control group. The agents were given for 25 times through intravenous injection in human hepatocellular carcinoma orthotopic implantation models. When the experiment was over, the volume and weight of tumor were measure, the inhibitory rate of tumor growth was calculate, and the pathomorphological change was examined. Alpha fetal protein (AFP), routine blood test and biochemical indexes were determined in three experiments respectively. Results The average volume and weight of tumor in IGF-1R ASODN group was significantly reduced as compared with those in NS control group. The inhibitory rate of tumor growth in 75 mg/(kg·day) IGF-1R APSODN group in three experiments were 76.36%, 71.81% and 78.81%, 55.65%, 61.74% and 63.58% in 50 mg/(kg·day) group, and 47.72%, 50.34% and 54.30% in 25 mg/(kg·day) group respectively. Large piece of necrosis and fibrosis could be seen under a light microscope in IGF-1R ASODN groups. AFP in IGF-1R ASODN groups was lower (P<0.05) than in NS control group, PLT in IGF-1R ASODN group [75 mg/(kg·day)] was lower than in NS control group (P<0.05), and GLU in IGF-1R ASODN group [75 mg/(kg·day) and 50 mg/(kg·day)] was higher than in normal control group (P<0.05). There was no significant difference between IGF-1R ASODN group and normal control group in other blood indicators. Conclusion IGF-1R ASODN is an anti-hepatoma biological agent with high efficiency and low side-effect. Key words: Carcinoma, hepatocellular; Insulin-like growth factor 1 receptor; Antisense oligdeoxynucleotides; Inhibitory rates of tumor growth

Induction of Oxidative Stress Through Inhibition of Thioredoxin Reductase 1 Is an Effective Therapeutic Approach for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide which lacks effective treatment. Cancer cells experience high levels of oxidative stress due to increased generation of reactive oxygen species (ROS). Increased antioxidant-producing capacity is therefore found in cancer cells to counteract oxidative stress. The thioredoxin system is a ubiquitous mammalian antioxidant system which scavenges ROS, and we demonstrate that it is vital for HCC growth as it maintains intracellular reduction-oxidation (redox) homeostasis. Transcriptome sequencing in human HCC samples revealed significant overexpression of thioredoxin reductase 1 (TXNRD1), the cytosolic subunit and key enzyme of the thioredoxin system, with significant correlations to poorer clinicopathological features and patient survival. Driven by the transcriptional activation of nuclear factor (erythroid-derived 2)-like 2, the master protector against oxidative stress, TXNRD1 counteracts intracellular ROS produced in human HCC. Inhibition of TXNRD1 through genetic inhibition hindered the proliferation of HCC cells and induced apoptosis in vitro. Administration of the pharmacological TXNRD1 inhibitor auranofin (AUR) effectively suppressed the growth of HCC tumors induced using the hydrodynamic tail vein injection and orthotopic implantation models in vivo. Furthermore, AUR sensitized HCC cells toward the conventional therapeutic sorafenib. Conclusion: Our study highlights the reliance of HCC cells on antioxidants for redox homeostasis and growth advantage; targeting TXNRD1 resulted in dramatic accumulation of ROS, which was found to be an effective approach for the suppression of HCC tumor growth.

Long noncoding RNA HOXD-AS2 regulates cell cycle to promote glioma progression.

Now, numerous exciting findings have been found that long noncoding RNAs (lncRNAs) play a vital role in cancer malignant progression. However, their potential involvement in glioma is not well understood. Here, we performed a high-throughput microarray to detect the lncRNA expression profiles between glioma cell lines and normal astrocyte cell lines. HOXD-AS2 was increased in glioma cells and it was associated with glioma grade and poor prognosis. Loss of HOXD-AS2 can inhibit glioma cell growth by inducing cell-cycle G1 arrest in vitro. The proliferation of glioma was inhibited followed by knocking down the expression of HOXD-AS2 not only in subcutaneous injection model but also in orthotopic implantation model. These findings indicate that HOXD-AS2 promotes the glioma progression and may serve as a potential target for glioma diagnosis and therapy.

The antitumour growth and antiangiogenesis effects of xanthatin in murine glioma dynamically evaluated by dynamic contrast-enhanced magnetic resonance imaging.

To investigate the suppressive effects of xanthatin on glioma growth in a nude mouse xenograft model and rat orthotopic implantation model using magnetic resonance imaging (MRI) to dynamically monitor the antitumour growth and antiangiogenesis effects of xanthatin. The nude mouse xenograft tumour model and rat orthotopic implantation model were established to observe the antitumour effects of xanthatin in vivo. In the rat orthotopic implanted tumour model, MRI scanning was used to dynamically monitor the antitumour growth effect and evaluate the antiangiogenesis effect of xanthatin. We found that xanthatin at a dose of 0.4mg/10g dramatically decreased the growth of xenograft tumours in nude mice. The antiangiogenesis effect of xanthatin C6 glioma was evaluated by dynamic contrast-enhanced (DCE) MRI via comparison of the volume transfer constant (Ktrans ) value, a parameter that reflects vessel permeability. We found that xanthatin at the doses of 8 and 16mg/kg significantly decreased the Ktrans value, which suggests that xanthatin has antiangiogenesis effects. These data demonstrate the suppressive effects of xanthatin on C6 glioma occur via antiangiogenesis. Meanwhile, this study also provides evidence for the application of quantitative parameters of DCE-MRI for dynamically evaluating the growth and angiogenesis of intracranial tumours and for experimental and clinical research.

Abstract 2415: HEY1 counteracts hypoxia-induced oxidative stress via transcriptionally repressing PINK1 in hepatocellular carcinoma

Abstract Background and Objective: Excessive accumulation of oxidative stress/reactive oxygen species (ROS) can be harmful to cancer cells. Hypoxia or O2 deprivation, which is commonly found in hepatocellular carcinoma (HCC), is a crucial factor that contributes to elevated ROS level in HCC cells as hypoxia causes inefficient transfer of electrons in the mitochondria. To survive, HCC cells need to devise strategies to counteract and balance hypoxia-induced oxidative stress. While it is known that hypoxia inducible factors (HIFs) are essential to metabolic reprogramming in HCC cells under hypoxia, there are significant gaps in knowledge about underlying mechanisms and transcriptional targets of HIFs. Experimental Procedures: Gene profiling of HCC cell lines (exposed to 20% and 1% O2) was analyzed by transcriptome sequencing to identify novel candidate responsible for counteracting hypoxia-induced oxidative stress. ShRNA-mediated gene silencing and gene activation by CRISPR-dCas9 system were used to modify transcriptional expression of HEY1 for different functional assays. Transmission electron microscopy was used to visualize the mitochondrial structure. Orthotopic and subcutaneous HCC implantation models were used to evaluate the role of HEY1 in HCC progression. Transcriptome sequencing and ChIP assay were performed to identify novel transcriptional targets of HEY1. Results: We showed that transcriptional repressor HEY1 was induced under hypoxia and directly regulated by HIF-1α. Overexpression of HEY1 was associated with poor overall survival in HCC patients. Importantly, we identified PINK1 as a novel repression target of HEY1. PINK1 is known to protect cells against mitochondrial dysfunction. We demonstrated that HEY1 actively repressed PINK1 and downregulation of PINK1 led to loss of mitochondrial mass and impaired mitochondrial cristae formation, subsequently decreasing intracellular ROS level. Downregulation of PINK also associated with poor overall survival and decrease-free in HCC patients. Genetic ablation of HEY1 in HCC cells profoundly reduced tumor growth and lung metastasis while genetic ablation of PINK1 in HCC cells reversely promoted HCC growth. Strikingly, HEY1 and PINK1 expressions reversely correlated in human HCC tissues. Conclusion: This study unprecedentedly identifies an upstream regulatory mechanism of PINK1, which controls the oxidative stress in HCC cells. It also reveals a novel molecular mechanism by which ablation of HEY1 leads to elevation of oxidative stress, making HCC cells more vulnerable. Targeting HEY1 represents an attractive therapeutic approach against HCC. Citation Format: David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Dicky Cheuk-Ting Law, Vincent Wai-Hin Yuen, Larry Lai Wei, Hui-Yu Koh, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. HEY1 counteracts hypoxia-induced oxidative stress via transcriptionally repressing PINK1 in hepatocellular carcinoma [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 2415.

MiR-1290 promotes proliferation, migration, and invasion of glioma cells by targeting LHX6.

To investigate the interaction of miR-1290 and LHX6 in gliomas, and their influence on the propagation and metastasis of glioma cells. The differential expression of miR-1290 in glioma cells was identified by chip screening. The expression level of miR-1290 and LHX6 were determined by qRT-PCR and Western blot. The influence of miR-1290 on propagation of glioma cells were analyzed by MTT assay, EdU incorporation, and colony formation, while the impact of miR-1290 on metastasis was assessed by transwell assay. The relationship between LHX6 and miR-1290 was testified by luciferase reporter assay. The gliomas orthotopic implantation model of nude mice was established to investigate the influence of miR-1290 and LHX6 on tumor growth. Tumor volumes were evaluated by photon density, and the expression of Ki67 protein was analyzed by immunohistochemistry. MiR-1290 presented a higher expression in glioma cells and tissues. MiR-1290 overexpression significantly promoted propagation and metastasis of glioma cells, while miR-1290 knockdown inhibited glioma development. MiR-1290 suppressed LHX6 expression, facilitating development of glioma cells. The orthotopic implantation model showed that miR-1290 overexpression promoted tumor growth while LHX6 overexpression inhibited it. MiR-1290 could promote glioma cell propagation and metastasis by inhibiting LHX6.

Nickel chloride administration prevents the growth of oral squamous cell carcinoma.

The effect of NiCl2 on oral squamous cell carcinoma-derived cell line HSC3 was examined. Incubation with 1 mM NiCl2 significantly reduced the expression of MMPs at mRNA and protein levels. The in vivo orthotopic implantation model was established by injecting highly metastatic subcell line HSC3-M3 to nude mouse tongue. After 1 week of injection, mice were fed with or without 1 mM NiCl2-containing water for two to three weeks. Immunohistochamical examination revealed that MMP9 expression was drastically reduced in NiCl2-fed mice. By CT images, cancer mass was observed as a translucent area in control mice. In NiCl2-fed mice, much highly translucent area was observed within the translucent area. Histologically, this area corresponded to the necrotic area in the tumor mass. Real-time PCR analysis revealed the reduced expression of angiogenic factors such as IL-8 and VEGF mRNA in NiCl2-fed mice. To further examine the effect of NiCl2 on metastasis, human β-globin gene expression in regional lymphnodes was compared. The β-globin gene was totaly absent in NiCl2-fed mice. Moreover, various cancer metastasis-related genes were inhibited in NiCl2-fed mice by PCR array analysis. The results indicated that NiCl2 might be a promising new anti-cancer therapeutics for the oral cancer treatment.

Feiji Recipe inhibits the growth of lung cancer by modulating T-cell immunity through indoleamine-2,3-dioxygenase pathway in an orthotopic implantation model

ObjectiveEscape from the body’s immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe. MethodsAn orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography. ResultsCompared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4+CD25+ T-cells and Foxp3+ T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells. ConclusionThe molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

FF-10502, an Antimetabolite with Novel Activity on Dormant Cells, Is Superior to Gemcitabine for Targeting Pancreatic Cancer Cells.

In this paper, we report that 1-(2-deoxy-2-fluoro-4-thio-β-d-arabinofuranosyl) cytosine (FF-10502), a pyrimidine nucleoside antimetabolite with a chemical structure similar to gemcitabine, shows beneficial anticancer activity via a novel mechanism of action on dormant cells. The growth inhibition of pancreatic cancer cell lines by FF-10502 (IC50, 60-330 nM) was moderately weaker than that by gemcitabine in vitro. In contrast, an in vivo orthotopic implantation model in mice with established human pancreatic cancer cell line, SUIT-2, revealed no mortality with FF-10502 intravenous treatment, which was related to regression of implanted tumor and little metastasis, whereas 75% of the mice treated with gemcitabine died by day 128. Two in vivo patient-derived xenograft models with gemcitabine-resistant pancreatic cancer cells also demonstrated complete tumor growth suppression with FF-10502, but only partial inhibition with gemcitabine. We also investigated the mechanism of action of FF-10502 by using dormant cancer cells, which are reportedly involved in the development of resistance to chemotherapy. In vitro serum starvation-induced dormant SUIT-2 cells developed resistance to gemcitabine even in combination with DNA damage inducers (DDIs; H2O2, cisplatin, and temozolomide). Interestingly, FF-10502 in combination with DDIs significantly induced concentration-dependent cell death in accordance with enhanced DNA damage. FF-10502 was far more potent than gemcitabine in inhibiting DNA polymerase β, which may explain the difference in dormant cell injury, although further investigations for direct evidences are necessary. In conclusion, our study demonstrated the beneficial antitumor effects of FF-10502 in clinically relevant in vivo models, and suggests the importance of preventing DNA repair unlike gemcitabine.

Abstract P5-05-06: Development of advanced pre-clinical in vivo models of metastatic breast cancer

Abstract Backgrounds: The fact that we continue to lose 40,000 women with breast cancer every year in the US despite the recent advance in basic research clearly demonstrate disconnect in translation of basic research findings to clinic. This is largely due to lack of appropriate animal model that mimic clinical conditions for preclinical studies that result in high failure rate of clinical trials. To date, we had established many syngeneic mouse models, which are not free from limitations; 1) few clinically relevant animal models with bone metastasis have been established, 2) syngeneic mouse model cannot address human cancer genomics and tumor heterogeneity. Patient-Derived Xenograft (PDX) model has emerged as pre-clinical model to address these issues, however, it suffers low tumor take rate of around 20-40%, and lack metastatic model. Here, we describe development of orthotopic implantation, and bone and liver metastatic breast cancer mouse models to overcome these limitations. Methods: 1) 4T1.2-luc3 cells that has metastatic potential to the bone were orthotropically inoculated as a syngeneic mouse model, imaged with IVIS and MRI. 2) Patient tumor tissues of 1mm(3) were implanted surgically into dorsal subcutaneous space (SQ), or orthotropically into mammary fat pat #2 and #4 (MFP). Results: 1) We established a syngeneic breast cancer bone metastasis model. Primary tumors were surgically resected days after 4T1.2-luc3 cells were orthotopically implanted under direct vision. Removal of primary tumor allowed bioluminescent visualization and quantification of bone metastasis by IVIS. We found that MRI was effective in evaluating bone metastasis and bone related events in these mice. MRI allows differentiation of bone metastasis from metastasis to the surrounding organs with bone destruction image, whereas conventional bioluminescence imaging shows only existence of cancer cells. 2) The overall tumor take rate of the tumor in PDX model was 46.0% (74/161 implantation site). Take rate from triple-negative breast cancer tumors was 56.1% (74/132), on the other hand, that from ER positive tumors was 0% (0/39). Tumor take rate was significantly better in MFP implantation than SQ (39.5%, 30/76 vs 51.2%, 44/85, p&amp;lt;0.01). Tumor weight were significantly heavier in MFP compared to SQ (0.072g vs 0.328g, p&amp;lt;0.00001). With more passage, the difference in tumor weight between SQ and MFP was significantly increased(p&amp;lt;0.0001). Finally, we developed a PDX breast cancer liver metastasis model by surgically implanting tissue fragment into liver using direct vision technique. We found MRI to be very useful as a living imaging modality to evaluate cancer progression in the deeply located metastatic sites of PDX models. Conclusions: We have established orthotropic syngeneic breast cancer bone metastasis model as well as improved breast cancer PDX model with synchronous liver metastasis utilizing MRI. Our novel models could be powerful tools for preclinical studies. Citation Format: Okano M, Kawaguchi T, Okano I, Katsuta E, Takabe K. Development of advanced pre-clinical in vivo models of metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-06.

Bone regeneration capacity of magnesium phosphate cements in a large animal model

Magnesium phosphate minerals have captured increasing attention during the past years as suitable alternatives for calcium phosphate bone replacement materials. Here, we investigated the degradation and bone regeneration capacity of experimental struvite (MgNH4PO4·6H2O) forming magnesium phosphate cements in two different orthotopic ovine implantation models. Cements formed at powder to liquid ratios (PLR) of 2.0 and 3.0 g ml−1 were implanted into trabecular bone using a non-load-bearing femoral drill-hole model and a load-bearing tibial defect model. After 4, 7 and 10 months the implants were retrieved and cement degradation and new bone formation was analyzed by micro-computed tomography (µCT) and histomorphometry. The results showed cement degradation in concert with new bone formation at both defect locations. Both cements were almost completely degraded after 10 months. The struvite cement formed with a PLR of 2.0 g ml−1 exhibited a slightly accelerated degradation kinetics compared to the cement with a PLR of 3.0 g ml−1. Tartrat-resistant acid phosphatase (TRAP) staining indicated osteoclastic resorption at the cement surface. Energy dispersive X-ray analysis (EDX) revealed that small residual cement particles were mostly accumulated in the bone marrow in between newly formed bone trabeculae. Mechanical loading did not significantly increase bone formation associated with cement degradation. Concluding, struvite-forming cements might be promising bone replacement materials due to their good degradation which is coupled with new bone formation. Statement of SignificanceRecently, the interest in magnesium phosphate cements (MPC) for bone substitution increased, as they exhibit high initial strength, comparably elevated degradation potential and the release of valuable magnesium ions. However, only few in vivo studies, mostly including non-load-bearing defects in small animals, have been performed to analyze the degradation and regeneration capability of MPC derived compounds. The present study examined the in vivo behavior of magnesiumammoniumphosphate hexahydrate (struvite) implants with different porosity in both mechanically loaded and non-loaded defects of merino sheep. For the first time, the effect of mechanical stimuli on the biological outcome of this clinically relevant replacement material is shown and directly compared to the conventional unloaded defect situation in a large animal model.

Repression of WT1-Mediated LEF1 Transcription by Mangiferin Governs β-Catenin-Independent Wnt Signalling Inactivation in Hepatocellular Carcinoma

Background/Aims: The development of hepatocellular carcinoma (HCC) is a complex process which involves deregulation of multiple signalling pathways. The hyper-activation of Wnt signalling promotes sustained expansion, invasion, and neovascularization of HCC. Mangiferin, a natural small molecule present in Mangifera indica L. has been shown to inactivate β-catenin, which is an indispensable regulator in Wnt pathway. Our study aimed to determine whether mangiferin has any inhibitory effect on HCC and examine how it modulates Wnt signalling. Methods: The tumour inhibitory effect of mangiferin was examined by in vitro cellular models and an in vivo orthotopic HCC implantation model. The genes responsible for mangiferin-mediated anti-HCC were delineated by polymerase chain reaction (PCR) microarray. The expression of target genes was further determined by quantitative PCR and immuno-blotting assays. The binding capacity of Wilms’ tumour 1 (WT1) to the lymphoid enhancer-binding factor 1 (LEF1) promoter was confirmed by chromatin immunoprecipitation-qPCR. Results: Oral administration of mangiferin inhibited orthotopic tumour growth. Cellular investigations confirmed the dose-dependent inhibition of mangiferin on HCC expansion and invasion. PCR array combined with Gene Ontology analysis revealed that the Wnt pathway was the predominant target of mangiferin and LEF1 was the most reduced gene in the Wnt pathway. Overexpression of LEF1 diminished repression of Wnt signalling and reduced proliferation activity in mangiferin-treated HCC cells. The mangiferin-mediated down-regulation of LEF1 was independent of β-catenin but associated with WT1 protein. WT1 knock-in in HCC cells further enhanced LEF1 expression. Chromatin immunoprecipitation assays revealed that the mangiferin induced repression of LEF1 was associated with decreased occupancy of WT1 on the LEF1 promoter. Conclusion: Our study identifies a novel mechanism of hepatocellular carcinoma inhibition through β-catenin-independent Wnt signalling, which is regulated by WT1-associated LEF1 repression. The study also highlights mangiferin as a promising Wnt inhibitor for HCC treatment.

Experimental evidence of good efficacy and reduced toxicity with peptide-doxorubicin to treat gastric cancer.

BackgroundTo compare the efficacy and toxicity of peptide-doxorubicin (PDOX) and doxorubicin (DOX) on nude mice models of human gastric cancer.ResultsBoth PDOX and DOX could significantly inhibit tumor growth compared with Control (P < 0.05) in both subcutaneous and orthotopic models. Animal survival was much better in PDOX group than DOX group. In peripheral blood test, PDOX group had significantly higher levels of platelets than the Control (P < 0.05), and lymphocyte lower than Control (P < 0.05). There were no significant differences on liver, kidney and cardiac function parameters among three groups (P > 0.05). Immunohistochemistry showed that treatment groups had much higher Tunel than Control (P < 0.05), and PDOX had significantly lower Ki-67 than doxorubicin and Control group (P < 0.01). Western blotting showed that PDOX caused much higher expressions of P53, P21, Aparf-1, pro- and cleaved-caspase 3, compared with DOX.ConclusionCompared with DOX, PDOX has increased effects but much decreased toxicity in treating animal model of gastric cancer.Materials and MethodsAnimals in subcutaneous model were randomized into Control, doxorubicin, PDOX-L, PDOX-M, and PDOX-H groups. Animals in surgical orthotopic implantation model were randomized into Control, doxorubicin and, peptide-doxorubicin groups. The animals were treated, monitored and examined following a set protocol.

The role of EpCAM in tumor progression and the clinical prognosis of endometrial carcinoma

ObjectiveEpCAM is a transmembrane glycoprotein that functions as an epithelial marker in endometrial tissues. However, the correlation between EpCAM and endometrial carcinoma (EC) is not clear. MethodsThis study investigated the association between EpCAM and EC. Immunohistochemistry staining and bioinformatics analysis disclosed the clinical importance of low EpCAM expression. The migratory ability of cells expressing low EpCAM levels was studied in transwell invasion assays in vitro and an orthotopic intra-uterine tumor injection model in vivo. The Connectivity MAP was used to identify drugs that effectively inhibit cells with low EpCAM expression. ResultsAccording to immunohistochemistry analysis results, low EpCAM expression was associated with an advanced stage and lymph node metastasis in patients with endometrioid EC, and high EpCAM expression favored survival. EpCAM silencing promoted cell invasion, and EpCAM re-expression in EpCAM-silenced EC cells attenuated their invasiveness. EpCAM suppression in an orthotopic uterine implantation model promoted the lymph node metastasis of EC cells. According to quantitative PCR and promoter reporter analyses, estrogen receptor alpha signaling regulated EpCAM expression by enhancing its promoter activity. As shown in the Connectivity MAP analysis, transamin inhibited the invasiveness of EpCAM-silenced EC cells. ConclusionsThe loss of EpCAM may increase the malignancy of EC, and these findings provide new insights into the prognostic role of EpCAM in patients with EC.

MicroRNA-378 enhances radiation response in ectopic and orthotopic implantation models of glioblastoma.

Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor, which is virtually incurable due to its therapeutic resistance to radiation and chemotherapy. To develop novel therapeutic approaches for treatment of GBM, we examined the role of miR-378 on tumor growth, angiogenesis, and radiation response in ectopic and orthotopic U87 glioblastoma models. Cell and tumor growth rates, in vitro and in vivo radiation sensitivities, and tumor vascular density were evaluated in U87-GFP and U87-miR-378 tumor lines. Ectopic tumor response to radiation was evaluated under normal blood flow and clamp hypoxic conditions. Results show that in vitro, miR-378 expression moderately increased cell growth rate and plating efficiency, but did not alter radiation sensitivity. U87-miR-378 tumors exhibited a higher transplantation take rate than U87-GFP tumors. In vivo, under oxygenated condition, subcutaneous U87-miR-378 tumors receiving 25Gy showed a tendency for longer tumor growth delay (TGD) than control U87-GFP tumors. In contrast, under hypoxic condition, U87-miR-378 xenografts exhibited substantially shorter TGD than U87-GFP tumors, indicating that under normal blood flow conditions, U87-miR-378 tumors were substantially more oxygenated than U87-GFP tumors. Intracranial multi-photon laser-scanning microscopy demonstrated increased vascular density of U87-miR-378 versus control U87-GFP tumors. Finally, miR-378 increased TGD following 12Gy irradiation in U87 intracranial xenografts, and significantly prolonged survival of U87-miR-378 tumor-bearing mice (P = 0.04). In conclusion, higher miR-378 expression in U87-miR-378 cells promotes tumor growth, angiogenesis, radiation-induced TGD, and prolongs survival of orthotopic tumor-bearing hosts. Regulation of VEGFR2 by miR-378 significantly increased vascular density and oxygenation in U87 xenografts.

De Novo Lipid Synthesis Facilitates Gemcitabine Resistance through Endoplasmic Reticulum Stress in Pancreatic Cancer.

Pancreatic adenocarcinoma is moderately responsive to gemcitabine-based chemotherapy, the most widely used single-agent therapy for pancreatic cancer. Although the prognosis in pancreatic cancer remains grim in part due to poor response to therapy, previous attempts at identifying and targeting the resistance mechanisms have not been very successful. By leveraging The Cancer Genome Atlas dataset, we identified lipid metabolism as the metabolic pathway that most significantly correlated with poor gemcitabine response in pancreatic cancer patients. Furthermore, we investigated the relationship between alterations in lipogenesis pathway and gemcitabine resistance by utilizing tissues from the genetically engineered mouse model and human pancreatic cancer patients. We observed a significant increase in fatty acid synthase (FASN) expression with increasing disease progression in spontaneous pancreatic cancer mouse model, and a correlation of high FASN expression with poor survival in patients and poor gemcitabine responsiveness in cell lines. We observed a synergistic effect of FASN inhibitors with gemcitabine in pancreatic cancer cells in culture and orthotopic implantation models. Combination of gemcitabine and the FASN inhibitor orlistat significantly diminished stemness, in part due to induction of endoplasmic reticulum (ER) stress that resulted in apoptosis. Moreover, direct induction of ER stress with thapsigargin caused a similar decrease in stemness and showed synergistic activity with gemcitabine. Our in vivo studies with orthotopic implantation models demonstrated a robust increase in gemcitabine responsiveness upon inhibition of fatty acid biosynthesis with orlistat. Altogether, we demonstrate that fatty acid biosynthesis pathway manipulation can help overcome the gemcitabine resistance in pancreatic cancer by regulating ER stress and stemness. Cancer Res; 77(20); 5503-17. ©2017 AACR.

Abstract 5609: Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that shows striking resistance to standard therapies. Immunotherapy is an exciting new treatment strategy for PDAC, but has yet to reproducibly demonstrate clinical efficacy. This finding may reflect poor effector T cell infiltration into PDAC or mechanisms of peripheral T cell tolerance. We hypothesize that successful immunotherapy in PDAC will require strategies that reverse elements of innate immunity that regulate T cell tolerance. This hypothesis is based on our prior studies in the KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mouse model of PDAC where macrophages residing outside of the tumor microenvironment were found to regulate the efficacy of a vaccine strategy involving the combination of chemotherapy and a CD40 agonist (FGK45). Specifically, we found that depletion of peripheral phagocytes using clodronate-encapsulated liposomes (CEL) was necessary for gemcitabine chemotherapy and a CD40 agonist to induce productive anti-tumor T cell immunity against spontaneously arising PDAC tumors in the KPC model. We now investigate the mechanism by which CEL-depleted phagocytes regulate T cell tolerance in PDAC using the KPC mouse model and an orthotopic implantation model. We found that CEL depletes macrophages in the spleen and abdominal lymph nodes, thereby reversing the pathologic accumulation of macrophages in these secondary lymphoid organs that is induced by PDAC tumors. CEL targeted two macrophage subsets in the lymph node, CD11b+CD169+F4/80neg subcapsular sinus macrophages (SSM) and CD11b+CD169+F4/80+ medullary sinus macrophages (MSM). These macrophage populations may be exposed to tumor-derived antigens and apoptotic cell vesicles entering the lymph node, and have been previously identified as central orchestrators of tolerance in autoimmune models. We found that both fluorescently-labeled liposomes and OVA-FITC peptide injected into the peritoneal cavity were specifically targeted to SSM and MSM with little uptake by dendritic cells (DC). In contrast, depletion of these first-line phagocytes by CEL shifted the cellular uptake of these model antigens increasing uptake by CD11b+CD11c+ DC which are more efficient in antigen presentation and show increased expression of co-stimulatory molecules after FGK45 treatment. Using an orthotopic model of PDAC, we then determined that CEL can enhance the T cell stimulatory capacity of gemcitabine chemotherapy in combination with FGK45 as seen by a significant increase in CD38, CD44, CD69 and PD-1 expression on CD4 and CD8 T cells in secondary lymphoid organs. Together, our findings suggest that tumor-derived antigens may be sequestered by tolerogenic macrophages in peritumoral lymph nodes that act to impair optimal induction of T cell responses. Thus, strategies aimed at disrupting the tolerogenic functions of these macrophages may offer a novel approach for improving immunotherapy in PDAC. Citation Format: Fee Bengsch, Anni Liu, Kathleen Graham, Gregory L. Beatty. Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer [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 5609. doi:10.1158/1538-7445.AM2017-5609