Peritoneal carcinomatosis of colorectal cancer is characterized by structural and functional reorganization of the tumor microenvironment inducing senescence and proliferation arrest in cancer cells.

Background : Peritoneal carcinomatosis (PC) is a terminal evolution from primary colorectal cancer (pCRC) associated with poor patient survival. Impact of the immune cell infiltrate on PC pathogenesis is unknown. Therefore, we characterized the immunological tumor microenvironment regarding proliferation, senescence and neovascularization. Methods : Formalin-fixed and paraffin-embedded (FFPE) tissue of PC and pCRC was examined by immunohistochemistry. Cells infiltrating resected tissue were isolated and analyzed by flow cytometry. PCR arrays detected the expression of genes relevant for helper T (TH) cell responses, like TH1, TH2 and TH17 response. Results : PC tumor cells demonstrate significantly lower proliferation rates than pCRC, but show significantly more senescence. PC is surrounded by significantly increased numbers of cytotoxic active Natural Killer (NK) cells, follicular helper T cells (TFH) and B cells, whereas pCRC shows more CD4+ TH cells, CD8+ cytotoxic T (TC) cells, eosinophilic granulocytes, TH17 and regulatory T (Treg) cells. PC is characterized by significantly increased interferon-γ (IFNγ), an upregulation of tumor necrosis factor (TNF) and the NK cell-regulating cytokine interleukin-15 (IL-15). An upregulation of angiogenesis-related genes, like vascular endothelial growth factor-A (VEGF-A), leads to severe neovascularization in PC. Correlations of PC results reveal that elevated numbers of interleukin-17 (IL-17) positive cells are associated with high cancer cell proliferation, whereas high numbers of IFNγ positive cells correlate with more tumor cells in senescence. Conclusion : The cellular immune reaction is modified during metastasis, inducing senescence in PC tumor cells. Immune surveillance in PC is facilitated by NK cells and high levels of IFNγ and TNF. Counteracting this effect, TFH and B cells combined with VEGF-A enhancement promote neovascularization in PC (Illustration 1). During metastasis from primary CRC to PC the immune cell infiltrate changes, accompanied by the induction of senescence in PC cancer cells (marked red): In pCRC, the antitumor immune response is facilitated by CD4+TH cells, CD8+TC cells and PRG2+ eosinophilic granulocytes. The premetastatic niche development is promoted by Treg cells and TH17 cells producing systemic factors like VEGF-A, TGF-ß and TNF. Along with TFH and B cells, as with a pro-tumor immune response, they support metastatic formation and lead to severe neovascularization in PC. This is counterbalanced by the IL-15-induced activation and proliferation of NK cells. The secreted cytokines IFNγ and TNF mediate immunosurveillance.

Targeting cMET with INC280 impairs tumour growth and improves efficacy of gemcitabine in a pancreatic cancer model.

BACKGROUND: Expression and activation of the cMET receptor have been implicated in tumor progression and resistance to chemotherapy in human pancreatic cancer. In this regard we assessed the effects of targeting cMET in pancreatic cancer models in vitro and in vivo. METHODS: Human (L3.6pl, BxP3, HPAF-II, MiaPaCa2) and murine (Panc02) pancreatic cancer cell lines, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) were used for the experiments. Furthermore, the human pancreatic cancer cell line MiaPaCa2 with acquired resistance to gemcitabine was employed (MiaPaCa2(G250)). For targeting the cMET receptor, the oral available, ATP-competitive inhibitor INC280 was used. Effects of cMET inhibition on cancer and stromal cells were determined by growth assays, western blotting, motility assays and ELISA. Moreover, orthotopic xenogeneic and syngeneic mouse (BALB-C nu/nu; C57BL/6) models were used to assess in vivo efficacy of targeting cMET alone and in combination with gemcitabine. RESULTS: Treatment with INC280 impairs activation of signaling intermediates in pancreatic cancer cells and ECs, particularly when cells were stimulated with hepatocyte growth factor (HGF). Moreover, motility of cancer cells and ECs in response to HGF was reduced upon treatment with INC280. Only minor effects on VSMCs were detected. Interestingly, MiaPaCa2(G250) showed an increase in cMET expression and cMET inhibition abrogated HGF-induced effects on growth, motility and signaling as well as DFX-hypoxia HIF-1alpha and MDR-1 expression in vitro. In vivo, therapy with INC280 alone led to inhibition of orthotopic tumor growth in xenogeneic and syngeneic models. Similar to in vitro results, cMET expression was increased upon treatment with gemcitabine, and combination of the cMET inhibitor with gemcitabine improved anti-neoplastic capacity in an orthotopic syngeneic model. Immunohistochemical analysis revealed a significant inhibition of tumor cell proliferation (Ki67) and tumor vascularization (CD31). Finally, combination of gemcitabine with INC280 significantly prolonged survival in the orthotopic syngeneic tumor model even when treatment with the cMET inhibitor was initiated at an advanced stage of disease. CONCLUSIONS: These data provide evidence that targeting cMET in combination with gemcitabine may be effective in human pancreatic cancer and warrants further clinical evaluation.

Transforming growth factor-beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer.

Pancreatic cancer is one of the most aggressive human cancers with a 5-year survival rate of <5%. Overexpression of transforming growth factor-beta 2 (TGF-ß2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF-ß2 mRNA and was successfully tested in a randomized, active-controlled phase IIb clinical study in patients with high-grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF-ß2 secretion in human pancreatic cell lines with an IC50 in the low µM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF-ß2-mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell-mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen.

Dual inhibition of Raf and VEGFR2 reduces growth and vascularization of hepatocellular carcinoma in an experimental model.

BACKGROUND AND AIMS: Activation of the mitogen-activated protein kinase-extracellular-signal-regulated kinase (ERK) pathways plays an important role in the progression of hepatocellular carcinoma (HCC). Importantly, Raf kinases are principal effectors within this oncogenic signaling cascade. We hypothesized that concomitant inhibition of Raf and vascular endothelial growth factor receptor 2 (VEGFR2) will affect tumor growth and angiogenesis of HCC. MATERIALS AND METHODS: Human HCC cell lines, endothelial cells (EC), and vascular smooth muscle cells (VSMC) were used. For blocking Raf kinase and VEGFR2, the small molecule inhibitor NVP-AAL881 (Novartis, USA) was used. Activation of signaling intermediates was assessed by Western blotting, and changes in cell motility were evaluated in migration assays. Effects of NVP-AAL881 on HCC growth were determined in a subcutaneous tumor model. RESULTS: NVP-AAL881 disrupted activation of ERK and STAT3 in HCC cells and reduced cancer cell motility. In addition, the migration of ECs and VSMC was also significantly impaired. In ECs, HCC-conditioned media-induced activation of STAT3 was diminished by NVP-AAL881 treatment. In vivo, NVP-AAL881 significantly reduced tumor growth, CD31-vessel area, and numbers of BrdU-positive proliferating tumor cells. CONCLUSIONS: Combined inhibition of Raf and VEGFR2 disrupts oncogenic signaling and efficiently reduces tumor growth and vascularization of HCC. Hence, this strategy could prove valuable for therapy of HCC.