Targeted TLR9 Agonist Elicits Effective Antitumor Immunity against Spontaneously Arising Breast Tumors.

Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.

Systemic delivery of a targeted synthetic immunostimulant transforms the immune landscape for effective tumor regression.

Promoting immune activation within the tumor microenvironment (TME) is a promising therapeutic strategy to reverse tumor immunosuppression and elicit anti-tumor immunity. To enable tumor-localized immunotherapy following intravenous administration, we chemically conjugated a polyspecific integrin-binding peptide (PIP) to an immunostimulant (Toll-like receptor 9 [TLR9] agonist: CpG) to generate a tumor-targeted immunomodulatory agent, referred to as PIP-CpG. We demonstrate that systemic delivery of PIP-CpG induces tumor regression and enhances therapeutic efficacy compared with untargeted CpG in aggressive murine breast and pancreatic cancer models. Furthermore, PIP-CpG transforms the immune-suppressive TME dominated by myeloid-derived suppressor cells into a lymphocyte-rich TME infiltrated with activated CD8+ T cells, CD4+ T cells, and B cells. Finally, we show that T cells are required for therapeutic efficacy and that PIP-CpG treatment generates tumor-specific CD8+ T cells. These data demonstrate that conjugation to a synthetic tumor-targeted peptide can improve the efficacy of systemically administered immunostimulants and lead to durable anti-tumor immune responses.

Acinar cell clonal expansion in pancreas homeostasis and carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths worldwide1. Studies in human tissues and in mouse models have suggested that for many cancers, stem cells sustain early mutations driving tumour development2,3. For the pancreas, however, mechanisms underlying cellular renewal and initiation of PDAC remain unresolved. Here, using lineage tracing from the endogenous telomerase reverse transcriptase (Tert) locus, we identify a rare TERT-positive subpopulation of pancreatic acinar cells dispersed throughout the exocrine compartment. During homeostasis, these TERThigh acinar cells renew the pancreas by forming expanding clones of acinar cells, whereas randomly marked acinar cells do not form these clones. Specific expression of mutant Kras in TERThigh acinar cells accelerates acinar clone formation and causes transdifferentiation to ductal pre-invasive pancreatic intraepithelial neoplasms by upregulating Ras-MAPK signalling and activating the downstream kinase ERK (phospho-ERK). In resected human pancreatic neoplasms, we find that foci of phospho-ERK-positive acinar cells are common and frequently contain activating KRAS mutations, suggesting that these acinar regions represent an early cancer precursor lesion. These data support a model in which rare TERThigh acinar cells may sustain KRAS mutations, driving acinar cell expansion and creating a field of aberrant cells initiating pancreatic tumorigenesis.

Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury.

Hepatocytes are replenished gradually during homeostasis and robustly after liver injury1, 2. In adults, new hepatocytes originate from the existing hepatocyte pool3-8, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases9-11. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERTHigh hepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERTHigh hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERTHigh hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERTHigh hepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a 'distributed model' of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.

BCL3 Reduces the Sterile Inflammatory Response in Pancreatic and Biliary Tissues.

BACKGROUND & AIMS: Under conditions of inflammation in the absence of micro-organisms (sterile inflammation), necrotic cells release damage-associated molecular patterns that bind to Toll-like receptors on immune cells to activate a signaling pathway that involves activation of IκB kinase and nuclear factor κB (NF-κB). Little is known about the mechanisms that control NF-κB activity during sterile inflammation. We analyzed the contribution of B-cell CLL/lymphoma 3 (BCL3), a transcription factor that associates with NF-κB, in control of sterile inflammation in the pancreas and biliary system of mice. METHODS: Acute pancreatitis (AP) was induced in C57BL/6 (control) and Bcl3(-/-) mice by intraperitoneal injection of cerulein or pancreatic infusion of sodium taurocholate. We also studied Mdr2(-/-) mice, which develop spontaneous biliary inflammation, as well as Bcl3(-/-)Mdr2(-/-) mice. We performed immunohistochemical analyses of inflamed and noninflamed regions of pancreatic tissue from patients with AP or primary sclerosing cholangitis (PSC), as well as from mice. Immune cells were characterized by fluorescence-activated cell sorting analysis. Control or Bcl3(-/-) mice were irradiated, injected with bone marrow from Bcl3(-/-) or control mice, and AP was induced. RESULTS: Pancreatic or biliary tissues from patients with AP or PSC had higher levels of BCL3 and phosphorylated RelA and IκBα in inflamed vs noninflamed regions. Levels of BCL3 were higher in pancreata from control mice given cerulein than from mice without AP, and were higher in biliary tissues from Mdr2(-/-) mice than from control mice. Bcl3(-/-) mice developed more severe AP after administration of cerulein or sodium taurocholate than control mice; pancreata from the Bcl3(-/-) mice with AP had greater numbers of macrophages, myeloid-derived suppressor cells, dendritic cells, and granulocytes than control mice with AP. Activation of NF-κB was significantly prolonged in Bcl3(-/-) mice with AP, compared with control mice with AP. Bcl3(-/-)Mdr2(-/-) mice developed more severe cholestasis and had increased markers of liver injury and increased proliferation of biliary epithelial cells and hepatocytes than Mdr2(-/-) mice. In experiments with bone marrow chimeras, expression of BCL3 by acinar cells, but not myeloid cells, was required for reduction of inflammation during development of AP. BCL3 inhibited ubiquitination and proteasome-mediated degradation of p50 homodimers, which prolonged binding of NF-κB heterodimers to DNA. CONCLUSIONS: BCL3 is up-regulated in inflamed pancreatic or biliary tissues from mice and patients with AP or cholangitis. Its production appears to reduce the inflammatory response in these tissues via blocking ubiquitination and proteasome-mediated degradation of p50 homodimers.

Interleukin-6 in inflammatory and malignant diseases of the pancreas.

Interleukin-6 is an important pro-inflammatory cytokine strongly linked to the most burdened exocrine pancreatic diseases including acute pancreatitis, chronic pancreatitis and pancreatic cancer. However, its role in all these diseases is versatile and not completely defined. Several studies provided accumulating evidence that IL-6 is mainly involved in the JAK/STAT pathway activation promoting acute and chronic pancreatitis disease aggravation as well as pancreatic cancer initiation and progression. This review will focus on recent studies illustrating the role of IL-6 in acute and chronic pancreatitis and pancreatic oncogenesis. Further, a short overview of indicated disease pathologies will be given and the impact of IL-6 in JAK/STAT pathway, persistent STAT3 activation and cancer immunotherapy will be discussed.

IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality.

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis-associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.

Deletion of IκBα activates RelA to reduce acute pancreatitis in mice through up-regulation of Spi2A.

BACKGROUND & AIMS: The transcription factor nuclear factor-κB (NF-κB) (a heterodimer of NF-κB1p50 and RelA) is activated rapidly in acute pancreatitis (AP). However, it is not clear whether NF-κB promotes or protects against AP. We used the NF-κB inhibitor protein, inhibitor of κB (IκB)α, to study the roles of NF-κB in the development of AP in mice. METHODS: IκBα or the combination of IκBα and RelA selectively were deleted from pancreas of mice using the Cre/locus of cross-over P strategy; cerulein or L-arginine were used to induce AP. We performed microarray analyses of the IκBα- and RelA-deficient pancreata. DNA from healthy individuals and patients with acute or chronic pancreatitis were analyzed for variants in coding regions of alpha-1-antichymotrypsin. RESULTS: Mice with pancreas-specific deletion of IκBα had constitutive activation of RelA and a gene expression profile consistent with NF-κB activation; development of AP in these mice was attenuated and trypsin activation was impaired. However, AP was fully induced in mice with pancreas-specific deletion of IκBα and RelA. By using genome-wide expression analysis, we identified a cluster of NF-κB-regulated genes that might protect against the development of AP. The serine protease inhibitor 2A (Spi2a) was highly up-regulated in IκBα-deficient mice. Lentiviral-mediated expression of Spi2A reduced the development of AP in C57BL/6 and RelA-deficient mice. However, we did not correlate any variants of alpha-1-antichymotrypsin, the human homologue of Spi2a, with acute or chronic pancreatitis. CONCLUSIONS: Pancreas-specific deletion of IκBα results in nuclear translocation of RelA and reduces AP induction and trypsin activation in mice after administration of cerulein or L-arginine. Constitutive activation of RelA up-regulates Spi2A, which protects mice against the development of AP.

Myeloid, but not pancreatic, RelA/p65 is required for fibrosis in a mouse model of chronic pancreatitis.

BACKGROUND & AIMS: Little is known about how transcription factors might regulate pathogenesis of chronic pancreatitis (CP). We analyzed the in vivo role of RelA/p65, a component of the transcription factor nuclear factor (NF)-κB, in different cell types during development of CP in mice. METHODS: RelA/p65 was functionally inactivated in the pancreas (relaΔpanc), in myeloid cells (relaΔmye), or both (relaΔpanc,Δmye) compartments using the Cre-loxP strategy. Experimental CP was induced with repetitive injections of cerulein over 6 weeks. Pancreata were investigated histologically and biochemically. We created an in vitro coculture assay of pancreatic stellate cells (PSC) and macrophages and performed gene arrays from pancreata and macrophages with functionally inactivated RelA/p65. Tissue samples from patients with CP were analyzed for matrix metalloproteinase (MMP) 10 expression. RESULTS: In contrast to their relaF/F littermates, relaΔpanc displayed typical signs of CP after long-term stimulation with cerulein. Numerous macrophages and activated α-smooth muscle actin (SMA)-positive PSCs were detected. Additional inactivation of RelA/p65 in myeloid cells (relaΔpanc,Δmye) attenuated fibrosis. In vitro, RelA/p65-deficient, lipopolysaccharide (LPS)-stimulated macrophages degraded fibronectin in cocultured PSCs. Using gene expression analysis, MMP-10 was identified as a candidate for this process. Recombinant MMP-10 degraded fibronectin in LPS-stimulated PSCs. In tissue samples from patients with CP, MMP-10 was up-regulated in myeloid cells. CONCLUSIONS: RelA/p65 functions in myeloid cells to promote pathogenesis of CP. In acinar cells, RelA/p65 protects against chronic inflammation, whereas myeloid RelA/p65 promotes fibrogenesis. In macrophage, MMP-10 functions as a RelA/p65-dependent, potentially antifibrogenic factor during progression of CP.