Research On Molecular Targeted Therapy Of Gastric Cancer Based On PDTX Models

Background:Gastric cancer (GC) is the one of the most commonly diagnosed cancers, and the second leading cause of cancer deaths worldwide. Despite improvements in surgery and chemotherapy, the prognosis of advanced gastric cancer remains poor, with a five-year survival rate nearly20%.Over the past decade, targeted therapies have greatly improved the outcome of a number of malignancies, including breast, colorectal and lung cancer, however, less progress has been made with regard to gastric cancer. One of the main obstacles hampering progress in anticancer drug development is a lack of clinic-relevant xenogaft models. Mouse xenografts derived from prolonged in vitro cultivated cells have been the standard toolkit for preclinical anti-cancer drugs evaluation for decades, however high failure rate of compounds entering clinical trials make us to suspect the clinical predictive power of the traditional tumor model system. However, prolonged in vitro culture and possible selection cause cell-implanted xenograft models to lose the original molecular characteristics and heterogeneity of primary tumors, which results in poor prediction of the clinical tumor’s drug response.In contrast to cell line-derived xenografts, patient derived tumor xenografts (PDTX) closely retain the histopathologic, genetic, and phenotypic features of the patients’ original tumors and could better predict drug efficacy in humans. In the last few years, PDTX models have been widely established in various tumor, including lung cancer, colorectal cancer, breast cancer, pancreatic cancer and gastric cancer. PDTX models is now becoming a powerful tool for the study of tumor biology and anticancer drugs evaluation.Approving Trastuzumab for HER2-positive GC patients represents a milestone in targeted gastric cancer therapy. Howerver, until now, only ramucirumab and apatinib have been approved for advanced gastric cancer as second-line treatment; however, the improved overall survival by targeted therapy is still limited. Therefore, developing new therapeutic approaches, particularly through the use of targeted therapeutic agents, is crucial for gastric cancer. Recently, members of RTK family,such as cMet,FGFR2and EGFR,have been intensely investigated in gastric cancer.Meanwhile, Many studies are now exploring the anti-tumor effect of relevant pathway inhibitors.Thus PDTX models of gastric cancer could be used as an ideal platform for screening of potential targeted agents, and provide more informative data for clinical drug efficacy testing.Section IBackground:Targeted therapies are emerging treatment options for gastric cancer (GC). Patient-derived tumor xenograft (PDTX) models of GC closely retain the features of the original clinical cancer, offering a powerful tool for preclinical drug efficacy testing. This study aimed to establish PDTX GC models, and explore therapeutics targeting Her2, cMet, and FGFR2, to further provide a basis for clinical drug efficacy testing.Methods:GC tissues from32patients were collected and implanted into immuno-deficient mice. Using immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH), protein levels and/or gene amplification of Her2, cMet and FGFR2in163GC patients were assessed. Finally, anti-tumor efficacy was tested in the PDTX models using targeted inhibitors.Results:A total of9passable PDTX models were successfully established. Of the163GC patients, HER2, cMet and FGFR2alterations were found in17(10.4%),32 (19.6%) and6(3.7%) individuals, and4(12.5%),8(25.0%) and1(3.1%) among the32xenograft donors, respectively. cMet gene amplification or protein overexpression (IHC3+) was associated with poor prognosis. Critozinib and ADZ4748ADZ4547exerted marked antitumor effects exclusively in PDTX models with cMet-(G30, G31) and FGFR2-(G03) amplification. Interestingly, increased antitumor activity was observed in G03(FGFR2-amplifed and cMet non-amplified but IHC [2+]) with simultaneous treatment of Critozinib and ADZ4547.Conclusion:These PDTX GC models provide an ideal platform for drug screening and evaluation. GC patients with positive cMet or FGFR2gene amplification may potentially benefit from cMet-or FGFR2-targeted therapies or combined targeted therapies.Section IIBackground:Aberrated activation of cMet in gastric cancer contributes to tumor growth, angiogenesis and metastasis. cMet-overexpressing gastric cancer has a poor prognosis because of high tumor metastasis and limited therapeutic options. Luteolin is a common dietary fiavonoid with antitumor properties. However, the antitumor effect of Luteolin on cMet-overexpressing gastric cancer remain unclear.Methods:Two cMet-overexpressing patient-derived human tumor xenograft (PDTX) models of gastric cancer were established, and treated with Luteolin or vehicle to evaluate the antitumor effects of Luteolin. Tumor specimens were subjected to H&E staining and immunohistochemistry. MKN45and SGC7901cells that show high cMet expression were treated with varying concentrations of Luteolin and evaluated by western blot, cell viability, apoptosis, migration, and invasion assays.Results:Luteolin inhibited the tumor growth in cMet-overexpressing PDTX models. Immunohistochemistry demonstrated that expression of cMet, MMP9and Ki-67were significantly down-regulated. Luteolin inhibited proliferation, promoted apoptosis and reduced the invasiveness of MKN45and SGC7901cells. Western blot revealed that Luteolin promoted the activation of apoptosis-related proteins, caspase-3and PARP-1, and down-regulated the invasion-associated protein, MMP9. Further studies demonstrated that Luteolin decreased the expression and phosphorylation of cMet, and downstream phosphorylation of AKT and ERK. In addition, Luteolin down-regulated phosphorylated AKT independently of cMet. Blocking AKT and/or ERK with the PI3K inhibitor, LY294002, or the ERK inhibitor, PD98059, induced down-regulation of MMP9and up-regulation of cleaved caspase-3and PARP-1, resembling the effects of Luteolin.Conclusions:Our findings, for the first time, demonstrate that Luteolin exerts marked antitumor effects in cMet-overexpressing PDTX models of gastric cancer, through a mechanism likely involving cMet/AKT/ERK signaling. These findings indicate that Luteolin may act as a potential therapeutic option for cMet-overexpressing gastric cancer. Section ⅢBackground:Alpha fetoprotein producing gastric cancer (AFPGC) is a rare type of gastric cancer with high liver metastasis incidence and poor prognosis. Less progress in targeted therapy has been made for AFPGC compared to common gastric cancer. This study aimed to explore the molecular segmentation of several known therapeutics Her2, cMet, EGFR, and VEGF in AFPGC patients and further evaluate potential targeted therapies in patient-derived tumor xenograft (PDTX) AFPGC models.Methods:Using immunohistochemistry (IHC) and FISH methods, we profiled protein expressions of Her2, cMet, EGFR, and VEGF and gene amplifications of HER2and MET in35AFPGCs and stage-matched70non-AFPGC samples. We established patient-derived tumor xenograft (PDTX) AFPGC models to evaluate targeted therapies’ efficacy. Results:We found cMet overexpression48.5%of the AFPGC group compared to20%in the non-AFPGC group. We found VEGF overexpression in71.4%of the AFPGC group compared to47.1%in the non-AFPGC group. We found cMet and VEGF co-overexpressed in42.8%of AFPGC patients, and they had a significant poorer survival rate compared to common AFPGC patients. There was no significant difference in EGFR and Her2expression between the AFPGC group and non-AFPGC group. We observed a significantly enhanced antitumor activity in cMet and VEGF co-overexpressed PDTX AFPGC models with simultaneous inhibition of c-Met (Crizotinib) and VEGF signaling (Bevacizumab).Conclusions:High frequency cMet and VEGF co-overexpression in AFPGC was associated with poor prognosis. This special AFPGC subtype may benefit from dual inhibition of cMet and VEGF signaling therapies.