Franco Novelli

IL-10 enhances CCL2 release and chemotaxis induced by CCL16 in human monocytes

CCL16 is a CC chemokine originally identified as a liver-expressed chemokine. Its expression has been detected in activated monocytes where it is up-regulated by stimulation with IL-10. This is in contrast with IL-10s inhibition of the expression of most chemokines. CCL16 is chemotactic for monocytes, lymphocyte and dendritic cells. We investigated whether CCL16 displays biological activities other than chemotaxis and whether IL-10 affects monocyte response to CCL16. We show that CCL16 induces the expression of CCL2 at the mRNA and protein level, but does not affect that of CCL5, CCL18 and proinflammatory cytokines. This effect was prevented by treatment with pertussis toxin and may thus be mediated by G-protein-coupled receptors. IL-10 markedly increased CCL2 production induced by CCL16, but suppressed that of CXCL8. It also enhanced the chemotactic response to CCL16. Addition of antibodies blocking CCR1, but not CCR8, prevented this enhanced chemotactic response and suggested that CCR1 is primarily involved. We propose that IL-10 modulates the effects of CCL16 on monocytes by increasing their CCR1-dependent response. The coordinated secretion of CCL16 and IL-10 may thus enhance monocyte infiltration.

Single kidney function: Effect of acute protein and water loading on microalbuminuria

The hyperfiltration induced by an acute response to an oral protein and water load was investigated to ascertain whether it can modify the urinary albumin excretion (UAE) in the microalbuminuric range by further increasing the glomerular filter permeability. To this end, six patients with a single kidney selected as having microalbuminuria on a regular diet without the clinical or laboratory data of overt renal disease and eight healthy subjects received a short-term protein and water load (150 g of meat-derived protein and 1 liter of water). In patients with one kidney, mean basal UAE values were significantly higher than in control subjects (p less than 0.006), whereas endogenous creatinine clearance values were only slightly lower (p greater than 0.05). One hour after the protein and water load, an abrupt increase in microalbuminuria levels was found in patients with one kidney and mean UAE values were significantly higher than in control subjects (p less than 0.002), whereas mean creatinine clearance values were significantly lower in patients than in control subjects (p less than 0.01). High UAE (p less than 0.002) and low creatinine clearance (p less than 0.002) values were maintained over the following four hours in patients with one kidney. These data suggest that in the single kidney with reduced renal functional reserve, an oral protein and water load magnifies the pre-existing loss of glomerular permselective properties due to chronic hyperfiltration as manifested by a further increase in microalbuminuria.

Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis.

Background:In pancreatic ductal adenocarcinoma (PDAC), fractalkine receptor CX3CR1 contributes to perineural invasion (PNI). We investigated whether CX3CR1 expression occurs early in PDAC and correlates with tumour features other than PNI.Methods:We studied CX3CR1 and CX3CL1 expression by immunohistochemistry in 104 human PDAC and coexisting Pancreatic Intraepithelial Neoplasia (PanIN), and in PdxCre/LSL-KrasG12D mouse model of PDAC. CX3CR1 expression in vitro was studied by a spheroid model, and in vivo by syngenic mouse graft of tumour cells.Results:In total, 56 (53.9%) PDAC expressed CX3CR1, 70 (67.3%) CX3CL1, and 45 (43.3%) both. CX3CR1 expression was independently associated with tumour glandular differentiation (P=0.005) and PNI (P=0.01). Pancreatic Intraepithelial Neoplasias were more frequently CX3CR1+ (80.3%, P<0.001) and CX3CL1+ (86.8%, P=0.002) than matched cancers. The survival of PDAC patients was better in those with CX3CR1+ tumour (P=0.05). Mouse PanINs were also CX3CR1+ and -CL1+. In vitro, cytokines significantly increased CX3CL1 but not CX3CR1 expression. Differently, CX3CR1 was upregulated in tumour spheroids, and in vivo only in well-differentiated tumours.Conclusion:Tumour differentiation, rather than inflammatory signalling, modulates CX3CR1 expression in PanINs and PDAC. CX3CR1 expression pattern suggests its early involvement in PDAC progression, outlining a potential target for interfering with the PanIN transition to invasive cancer.

A bioluminescent mouse model of proliferation to highlight early stages of pancreatic cancer: A suitable tool for preclinical studies.

Transgenic mouse models designed to recapitulate genetic and pathologic aspects of cancer are useful to study early stages of disease as well as its progression. Among several, two of the most sophisticated models for pancreatic ductal adenocarcinoma (PDAC) are the LSL-Kras(G12D/+);Pdx-1-Cre (KC) and LSL-Kras(G12D/+);LSL-Trp53(R172H/+);Pdx-1-Cre (KPC) mice, in which the Cre-recombinase regulated by a pancreas-specific promoter activates the expression of oncogenic Kras alone or in combination with a mutant p53, respectively. Non-invasive in vivo imaging offers a novel approach to preclinical studies introducing the possibility to investigate biological events in the spatio/temporal dimension. We recently developed a mouse model, MITO-Luc, engineered to express the luciferase reporter gene in cells undergoing active proliferation. In this model, proliferation events can be visualized non-invasively by bioluminescence imaging (BLI) in every body district in vivo. Here, we describe the development and characterization of MITO-Luc-KC- and -KPC mice. In these mice we have now the opportunity to follow PDAC evolution in the living animal in a time frame process. Moreover, by relating in vivo and ex vivo BLI and histopathological data we provide evidence that these mice could represents a suitable tool for pancreatic cancer preclinical studies. Our data also suggest that aberrant proliferation events take place early in pancreatic carcinogenesis, before tumour appearance.

CCL16 enhances the CD8+ and CD4+ T cell reactivity to human HER-2 elicited by dendritic cells loaded with rat ortholog HER-2.

T cells from HLA A2+ healthy donors were co-cultured with autologous dendritic cells (DC) loaded with apoptotic tumor cells expressing rat neu, and were induced to mature by tumor necrosis factor (TNF)α and interleukin (IL)-1β (mDC neu ) or by the CCL16 chemokine (CCL16/mDC neu ). Priming by CCL16/mDC neu induces a larger population of T cells that express cytoplasmatic interferon (IFN)γ, TNFα, perforin and granzyme B compared to those primed by mDCneu. T cells primed by CCL16/mDC neu release IFNγ in response to human HER-2+ cells and kill human HER-2+ target cells more efficiently than those primed by mDCneu. Our results show that both the loading of DC with xenogeneic rat neu and their maturation by CCL16 are two issues of critical importance for the elicitation of an effective response to human HER-2 in T cells from normal donors.

FAM49B, a novel regulator of mitochondrial function and integrity that suppresses tumor metastasis

Mitochondrial dysregulation plays a central role in cancers and drives reactive oxygen species (ROS)-dependent tumor progression. We investigated the pro-tumoral roles of mitochondrial dynamics and altered intracellular ROS levels in pancreatic ductal adenocarcinoma (PDAC). We identified ‘family with sequence similarity 49 member B’ (FAM49B) as a mitochondria-localized protein that regulates mitochondrial fission and cancer progression. Silencing FAM49B in PDAC cells resulted in increased fission and mitochondrial ROS generation, which enhanced PDAC cell proliferation and invasion. Notably, FAM49B expression levels in PDAC cells were downregulated by the tumor microenvironment. Overall, the results of this study show that FAM49B acts as a suppressor of cancer cell proliferation and invasion in PDAC by regulating tumor mitochondrial redox reactions and metabolism.

Abstract IA22: Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma

Ductal adenocarcinoma of the pancreas (PDAC) is a devastating disease that will afflict greater than 40,000 Americans. Less than 5% of these new pancreatic cancer patients will survive 5 years following diagnosis. PDAC is rarely detected in the early stages, with more than 80% of patients presenting with locally unresectable or metastatic disease at the time of diagnosis. As this form of cancer is also resistant to current cytotoxic therapies and ionizing radiation, novel therapeutic and diagnostic approaches are desperately needed to improve patient outcome. A characteristic feature of PDAC is the presence of an abundant, inflammatory infiltrate. In response to tumor-derived chemoattractants, we found that myeloid cells invade tumors where they promote immunosuppression, resulting in tumor growth and metastasis. We discovered that myeloid cell PI3-kinase gamma (PI3Kgamma) controls tumor immunosuppression, as PI3Kgamma signaling inhibits pro-inflammatory gene expression responses in macrophages, monocytes and granulocytes and promotes anti-inflammatory responses. Suppression of PI3Kgamma in mutant mice and in mice treated with pharmacologic inhibitors of PI3Kgamma promotes CD8+ T cell anti-tumor immune responses, leading to a 50% reduction in tumor growth and metastasis in orthotopic mouse models of pancreatic carcinoma and significant extension of survival in GEM models of PDAC (PDX1-cre; LSL-KrasG12D/+; LSL-Trp53 R172H/+). Furthermore, PI3Kgamma inhibition blocks macrophage stimulation of collagen deposition by fibroblasts in PDAC. We are exploring the combination of PI3kinase gamma inhibition with tumor cell and T cell targeted therapeutics could significantly reduce tumor growth and metastasis of PDAC. These approaches could improve patient outcomes by reducing tumor progression and preserving organ function by inhibiting desmoplasia. Citation Format: Megan Kaneda, Chanae Hardamon, Michael C. Schmid, Michael Bouvet, Franco Novelli, Emilio Hirsch, Andrew Lowy, Judith A. Varner. Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA22.