Background and aimsHelicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines.MethodsThree gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT.ResultsThe pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins.ConclusionThe upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric cancer, potentially through EMT, by indirectly increasing levels of soluble HB-EGF.

INTRODUCTION: Recent progress in the field of cancer immunotherapy have made it possible to translate several emerging immunostimulatory strategies, e.g. anti-CTLA-4, and anti-PD-1 into the clinic resulting in promising clinical benefits. In addition, a number of treatment strategies such as radiotherapy (RT) oncolytic viruses and certain chemotherapeutic agents e.g. Doxorubicin, Bortezomib and Mitoxantrone have been highlighted as potential inducers of immunogenic cell death through a mechanism resulting in the increased presentation of cell-associated antigens to CD4+ and CD8+ T lymphocytes by dendritic cells. Thus combination strategies of ICD inducers with immunotherapy (IT) could provide opportunities to harness the immune system to extend survival, even among metastatic and heavily pre-treated cancer patients, and may increase the efficacy of immunotherapy in those cancer types to be of a low immunogenic status. Here we compare the efficacy of immune checkpoint inhibitors in combination with documented ICD inducers to demonstrate an additive combination outcome in preclinical syngeneic models. EXPERIMENTAL PROCEDURES: Bioluminescent CT26 mouse colon cells, 4T1 mammary carcinoma cells or H22 hepatoma cells were implanted subcutaneously or orthotopically into BALB/c mice. Subcutaneous tumour growth was monitored by calliper measurement and bioluminescent imaging (BLI) was carried out to confirm orthotopic and/or metastatic growth. Established tumours were treated with immunotherapy in combination with chemotherapy, or hypofractionated image-guided micro-irradiation (IGMI) using the small animal research platform (SARRP; Xstrahl Ltd; body weight and clinical condition of mice were monitored daily. At termination the tumours were collected and assessed for immune cell infiltration and/or ICD markers by FACS and IHC. RESULTS: Response to treatment was evaluated by tumour growth inhibition or BLI following treatment of monotherapy or combinations of immunotherapy and ICD inducers (Oxaliplatin, Doxorubicin, and IGMI). Monotherapy with anti-CTLA4 exerted no statistically significant effect on primary or metastatic (4T1) tumour growth whereas ICD such as IR resulted in a modest tumour growth inhibition (TGI); When combined significant additive effect was observed (60% increase in TGI) on the primary tumour and reduction in tumour burden in the lungs indicating an abscopal effect. Details of modulation of immune cell infiltration and ICD markers observed in all models will be reported and correlated to response. CONCLUSIONS: Combination of immune checkpoint immunotherapy with a known ICD inducer (IGMI) resulted in an additive TGI in both CT26 and 4T1 models and effectively demonstrates their applicability for further exploring combination strategies involving immunotherapy. Citation Format: Andrew Mckenzie, Rajendra Kumari, Qian Shi, Nektaria Papadopoulou, Yinfei Yin, Simon Jiang, Jane Wrigley, Jason King, Neil Williams, Russell Garland. Immune competent syngeneic models demonstrate additive effects of combination strategies using checkpoint immunotherapy and inducers of immunogenic cell death (ICD). [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 3994.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients that have activating mutations in the EGFR gene are treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) e.g. Erlotinib (Tarceva®) and Gefitinib (Iressa®). Most NSCLC patients with activating EGFR mutations will respond to EGFR-TKIs; however, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of MET kinase and EMT conversion; however, additional routes of resistance are poorly defined. Using a novel patient-derived NSCLC xenograft model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. METHODS: LION102 is a NSCLC adenocarcinoma patient-derived xenograft (PDX) model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. Resistant models were generated in vivo through repeated cycling of treatment for up to 10 weeks with EGFR-TKI alone or in combination with a MET inhibitor. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing as well as for MET, AXL and HER2 over-expression and genomic amplification by quantitative PCR; Epithelial-to-Mesenchymal transition (EMT) was assessed by immunohistochemistry (IHC). RESULTS: Naïve LION102 PDX tumours exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p<0.0001). Following successive cycles of EGFR-TKI treatment in vivo several resistant subtypes were generated and characterized. CONCLUSIONS: EGFR-TKI resistant subtypes were generated in vivo from a proprietary patient-derived xenograft model (LION102) bearing an activating EGFR mutation (L858R) and characterised for their resistance mechanism. PDX models of resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR-TKIs. Citation Format: Andrew Mckenzie, Aaron Cranston, Phil Mallinder, Nektaria Papadopoulou, Simon Jiang, Kerry Moakes, Yinfei Yin, David Onion, Anna Grabowska, Martin Page, Rajendra Kumari. In vivo generation of EGFR-TKI resistance in a patient-derived xenograft (PDX) with an activating EGFR mutation (L858R), and molecular characterisation of resistance mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5651. doi:10.1158/1538-7445.AM2013-5651

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating NSCLC adenocarcinomas with targeted agents such as Erlotinib (Tarceva©) and Crizotinib (Xalkori©) few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of all NSCLC. Although the incidence of fibroblast growth factor receptor (FGFR) mutations in SCC is modest, amplification of FGFR is well documented and there are several first generation FGFR inhibitors currently in clinical trial. It is therefore essential to develop relevant in vivo patient-derived xenograft (PDX) models for the development and characterisation of new FGFR agents and/or combination strategies which may prolong benefit and delay the emergence of resistance. METHODS: LION137 is a novel NSCLC SCC patient-derived xenograft (PDX) model which is maintained subcutaneously in vivo admixed with a human stromal cell component. Cohorts of mice were dosed with AZD4547 alone or in combination with chemotherapy (CTx; parallel or serial dosing regimens). The extent of inhibition was measured over several in vivo passages and following cryopreservation/resuscitation. Response to treatment was evaluated by tumour growth inhibition and waterfall plots. Tumour material was characterised for 50 key oncogenes and mutations by Ion Torrent sequencing and FGFR1-4 expression and genomic amplification by quantitative PCR. Immunohistochemistry (IHC) was used to confirm SCC subtype, EMT status and FGFR expression. RESULTS: LION137 PDX tumours (SCC confirmed by IHC) exhibited reproducible sensitivity to AZD4547 alone (P<0.001) which was maintained both in continuous serial passage and following cryopreservation (at -196°C). FGFR expression and amplification and mutational analysis were assessed in naïve tumour material, following 10 weeks of continuous dosing and following outgrowth from CTX treatment and correlated with treatment response. CONCLUSIONS: LION137 is a novel patient-derived NSCLC SCC xenograft model with reproducible FGFRi-sensitivity which is directly relevant to the clinical setting. This model could be invaluable in assessing novel agents targeting the FGFR pathway and in the development of new combination strategies which may prevent or overcome resistance. Citation Format: Andrew Mckenzie, Aaron Cranston, Phil Mallinder, Nektaria Papadopoulou, Simon Jiang, Kerry Moakes, Yinfei Yin, David Onion, Anna Grabowska, Martin Page, Rajendra Kumari. In vivo response and molecular characterisation of a NSCLC squamous cell carcinoma PDX model exhibiting reproducible sensitivity to FGFR inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2013-342

Background: The rapid development of immuno-oncology (I/O) therapies for various cancer types has transformed the paradigm of cancer treatment from targeting the tumor to targeting the immune system, and now to combination strategies. Numerous Phase I-III trials with immunotherapeutics and combinations are being conducted; however, a lot of these are failing. More preclinical models are required to help investigate efficacy, uncover mechanisms of action, and to design more rational combination strategies. The most widely used models for immunotherapy evaluation are models with functional murine immunity such as syngeneic allograft models and genetically engineered mouse models (GEMMs). Syngeneic models are used in nearly all I/O therapy programs as these are well-characterized and straightforward to set-up as subcutaneous models. However, one of the major disadvantages of subcutaneous models is the lack of a clinically relevant tumor microenvironment (TME) including stromal cells, immune cells, extracellular matrix etc. The dynamic interplay of these cells drive alterations in cellular functions and impact study outcomes. Using bioluminescent imaging (BLI), we have developed and characterized a panel of orthotopic models where tumor cells are inoculated in a relevant organ-specific location to recapitulate the immune and stromal component interactions with the tumor, which can also facilitate metastatic spread. These models can be used to evaluate various therapies in both the orthotopic and metastatic settings. Method: A panel of syngeneic cell lines were labeled with bioluminescence by transducing the cell lines with a lentiviral vector carrying the firefly luciferase gene. This panel represents a diverse range of cancer types including liver (Hepa 1-6 and HT22), breast (4T1), colon (CT26.WT), brain (GL261), prostate (RM-1), and pancreas (Pan02). These bioluminescent cells were orthotopically implanted into immune competent mice. In-life tumor growth and metastasis were assessed using the IVIS® Spectrum In Vivo Imaging System. Tumor-infiltrating lymphocytes were assessed by flow cytometry (BD LSRFortessa™) and multiplex IHC. Results: Orthotopic implantation of syngeneic models was successfully demonstrated for all models as confirmed by in-life imaging and ex vivo imaging at termination. Baseline response towards various checkpoint inhibitors and chemotherapies were established. For the Hepa 1-6 liver model, response to an anti-PD-1 antibody was significant in both the subcutaneous and orthotopic setting. In contrast, the H22 liver model responded to both sorafenib and anti-mCTLA-4 antibody in the subcutaneous model but not in the orthotopic setting. In addition, anti-mCTLA-4 treatment led to more significant inhibition of pancreatic Pan02 tumor growth in the orthotopic model in comparison with its corresponding subcutaneous setting. Conclusion: Bioluminescent syngeneic models in the orthotopic setting provide a valuable tool for testing immunotherapies, with a more clinically relevant TME in comparison to the corresponding subcutaneous model. The establishment of a range of models covering diverse cancer types allows for a thorough interrogation of immunotherapeutic potential. Citation Format: Diandong Jiang, Bryan Miller, Yanrui Song, Rajendra Kumari, Annie An, Jie Cai, Davy Xuesong Ouyang, Henry Qixiang Li, Yinfei Yin. Characterization of a panel of orthotopic syngeneic models using bioluminescent imaging for the evaluation of immuno oncology therapeutics [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 2788.