Necuparanib, A Multitargeting Heparan Sulfate Mimetic, Targets Tumor and Stromal Compartments in Pancreatic Cancer.

Pancreatic cancer has an abysmal 5-year survival rate of 8%, making it a deadly disease with a need for novel therapies. Here we describe a multitargeting heparin-based mimetic, necuparanib, and its antitumor activity in both in vitro and in vivo models of pancreatic cancer. Necuparanib reduced tumor cell proliferation and invasion in a three-dimensional (3D) culture model; in vivo, it extended survival and reduced metastasis. Furthermore, proteomic analysis demonstrated that necuparanib altered the expression levels of multiple proteins involved in cancer-driving pathways including organ development, angiogenesis, proliferation, genomic stability, cellular energetics, and invasion and metastasis. One protein family known to be involved in invasion and metastasis and altered by necuparanib treatment was the matrix metalloprotease (MMP) family. Necuparanib reduced metalloproteinase 1 (MMP1) and increased tissue inhibitor of metalloproteinase 3 (TIMP3) protein levels and was found to increase RNA expression of TIMP3. MMP enzymatic activity was also found to be reduced in the 3D model. Finally, we confirmed necuparanib's in vivo activity by analyzing plasma samples of patients enrolled in a phase I/II study in patients with metastatic pancreatic cancer; treatment with necuparanib plus standard of care significantly increased TIMP3 plasma protein levels. Together, these results demonstrate necuparanib acts as a broad multitargeting therapeutic with in vitro and in vivo anti-invasive and antimetastatic activity.

Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.

Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to a broad range of DNA-damaging chemotherapeutics. We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16:4(n-3). Studies that used splenocytes from GPR120-deficient mice have confirmed this conclusion. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance-inducing lipid mediator, lysophosphatidylcholine(24:1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.-Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.

Intravital characterization of tumor cell migration in pancreatic cancer.

Curing pancreatic cancer is difficult as metastases often determine the poor clinical outcome. To gain more insight into the metastatic behavior of pancreatic cancer cells, we characterized migratory cells in primary pancreatic tumors using intravital microscopy. We visualized the migratory behavior of primary tumor cells of a genetically engineered pancreatic cancer mouse model and found that pancreatic tumor cells migrate with a mesenchymal morphology as single individual cells or collectively as a stream of non-cohesive single motile cells. These findings may improve our ability to conceive treatments that block metastatic behavior.

Increased Plasma Levels of Chemoresistance-Inducing Fatty Acid 16:4(n-3) After Consumption of Fish and Fish Oil.

IMPORTANCE: Our research group previously identified specific endogenous platinum-induced fatty acids (PIFAs) that, in picomolar quantities, activate splenic macrophages leading to resistance to chemotherapy in mouse models. Fish oil was shown to contain the PIFA 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) and when administered to mice neutralized chemotherapy activity. OBJECTIVE: Because patients with cancer frequently use fish oil supplements, we set out to determine exposure to 16:4(n-3) after intake of fish or fish oil. DESIGN, SETTING, AND PARTICIPANTS: (1) In November 2011, 400 patients with cancer undergoing treatment at the University Medical Center Utrecht were surveyed to determine their use of fish oil supplements; 118 patients responded to the questionnaire (30%); (2) pharmacokinetic analysis of the 16:4(n-3) content of 6 fish oils and 4 fishes was carried out; (3) from April through November 2012, a healthy volunteer study was performed to determine 16:4(n-3) plasma levels after intake of 3 different brands of fish oil or 4 different fish species. Thirty healthy volunteers were randomly selected for the fish oil study; 20 were randomly selected for the fish study. These studies were supported by preclinical tumor experiments in mice to determine chemoresistance conducted between September 2011 and December 2012. MAIN OUTCOMES AND MEASURES: (1) Rate of use of fish oil supplements among patients undergoing cancer treatment at our institution; (2) levels of 16:4(n-3) present in 3 brands of fish oil and 4 species of fish; and (3) plasma levels of 16:4(n-3) present in healthy volunteers after consuming fish oil or fish. RESULTS: Eleven percent of respondents reported using omega-3 supplements. All fish oils tested contained relevant amounts of 16:4(n-3), from 0.2 to 5.7 µM. Mouse experiments showed that addition of 1 µL of fish oil to cisplatin was sufficient to induce chemoresistance, treatment having no impact on the growth rate of tumors compared with vehicle-treated controls (estimated tumor volume difference, 44.1 mm3; P > .99). When the recommended daily amount of 10 mL of fish oil was administered to healthy volunteers, rises in plasma 16:4(n-3) levels were observed, reaching up to 20 times the baseline levels. Herring and mackerel contained high levels of 16:4(n-3) in contrast to salmon and tuna. Consumption of fish with high levels of 16:4(n-3) also resulted in elevated plasma levels of 16:4(n-3). CONCLUSIONS AND RELEVANCE: All tested fish oils and herring and mackerel fishes contained relevant levels of fatty acid 16:4(n-3), a fatty acid with chemotherapy-negating effects in preclinical models. After ingestion of these fish oils or fishes, 16:4(n-3) was rapidly taken up in the plasma of human volunteers. Until further data become available, fish oil and fish containing high levels of 16:4(n-3) may best be avoided on the days surrounding chemotherapy.

Lysophospholipids secreted by splenic macrophages induce chemotherapy resistance via interference with the DNA damage response.

Host responses to systemic anti-cancer treatment play important roles in the development of anti-cancer drug resistance. Here we show that F4/80(+)/CD11b(low) splenocytes mediate the resistance to DNA-damaging chemotherapeutics induced by two platinum-induced fatty acids (PIFAs), 12-S-keto-5,8,10-heptadecatrienoic acid and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) in xenograft mouse models. Splenectomy or depletion of splenic macrophages by liposomal clodronate protects against PIFA-induced chemoresistance. In addition, we find that 12-S-HHT, but not 16:4(n-3), functions via leukotriene B4 receptor 2 (BLT2). Genetic loss or chemical inhibition of BLT2 prevents 12-S-HHT-mediated resistance. Mass spectrometry analysis of conditioned medium derived from PIFA-stimulated splenic macrophages identifies several lysophosphatidylcholines as the resistance-inducing molecules. When comparing cisplatin and PIFA-treated tumours with cisplatin alone treated tumours we found overall less γH2AX, a measure for DNA damage. Taken together, we have identified an intricate network of lysophospholipid signalling by splenic macrophages that induces systemic chemoresistance in vivo via an altered DNA damage response.