A Listeria vaccine and depletion of T-regulatory cells activate immunity against early stage pancreatic intraepithelial neoplasms and prolong survival of mice.

BACKGROUND & AIMS: Premalignant lesions and early stage tumors contain immunosuppressive microenvironments that create barriers for cancer vaccines. Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which express an activated form of Kras in pancreatic tissues, develop pancreatic intraepithelial neoplasms (PanIN) that progress to pancreatic ductal adenocarcinoma (PDA). We used these mice to study immune suppression in PDA. METHODS: We immunized KPC and Kras(G12D/+);Pdx-1-Cre mice with attenuated intracellular Listeria monocytogenes (which induces CD4(+) and CD8(+) T-cell immunity) engineered to express Kras(G12D) (LM-Kras). The vaccine was given alone or in sequence with an anti-CD25 antibody (PC61) and cyclophosphamide to deplete T-regulatory (Treg) cells. Survival times were measured; pancreatic and spleen tissues were collected and analyzed by histologic, flow cytometry, and immunohistochemical analyses. RESULTS: Interferon γ-mediated, CD8(+) T-cell responses were observed in KPC and Kras(G12D/+);Pdx-1-Cre mice given LM-Kras, but not in unvaccinated mice. Administration of LM-Kras to KPC mice 4-6 weeks old (with early stage PanINs), depleted of Treg cells, significantly prolonged survival and reduced PanIN progression (median survival, 265 days), compared with unvaccinated mice (median survival, 150 days; P = .002), mice given only LM-Kras (median survival, 150 days; P = .050), and unvaccinated mice depleted of Treg cells (median survival, 170 days; P = .048). In 8- to 12-week-old mice (with late-stage PanINs), LM-Kras, alone or in combination with Treg cell depletion, did not increase survival time or slow PanIN progression. The combination of LM-Kras and Treg cell depletion reduced numbers of Foxp3(+)CD4(+) T cells in pancreatic lymph nodes, increased numbers of CD4(+) T cells that secrete interleukin 17 and interferon γ, and caused CD11b(+)Gr1(+) cells in the pancreas to acquire an immunostimulatory phenotype. CONCLUSIONS: Immunization of KPC mice with Listeria monocytogenes engineered to express Kras(G12D), along with depletion of Treg cells, reduces progression of early stage, but not late-stage, PanINs. This approach increases infiltration of the lesion with inflammatory cells. It might be possible to design immunotherapies against premalignant pancreatic lesions to slow or prevent progression to PDA.

CD8⁺ Foxp3⁺ tumor infiltrating lymphocytes accumulate in the context of an effective anti-tumor response.

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3-expressing CD8(+) T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti-tumor response. CD8(+) Foxp3(+) T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM-CSF secreting HER-2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen-specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3(+) and Foxp3(-) CD8(+) T cells have similar IFN-γ production and antigen-specific degranulation after stimulation with RNEU(420-429) , the immunodominant HER-2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU((420-429)) -specific effector T cells into neu-N mice (a model that results in immune tolerance to neu), confirm that CD8(+) Foxp3(+) T cells are present in tumors only if there is an existing pool of tumor-rejecting effector T cells. CD8(+) Foxp3(+) TILs mark the presence of tumor-rejecting antigen-specific T cells and their accumulation serves as a marker for an effective T cell response.

Semaphorin 3D autocrine signaling mediates the metastatic role of annexin A2 in pancreatic cancer.

Most patients with pancreatic ductal adenocarcinoma (PDA) present with metastatic disease at the time of diagnosis or will recur with metastases after surgical treatment. Semaphorin-plexin signaling mediates the migration of neuronal axons during development and of blood vessels during angiogenesis. The expression of the gene encoding semaphorin 3D (Sema3D) is increased in PDA tumors, and the presence of antibodies against the pleiotropic protein annexin A2 (AnxA2) in the sera of some patients after surgical resection of PDA is associated with longer recurrence-free survival. By knocking out AnxA2 in a transgenic mouse model of PDA (KPC) that recapitulates the progression of human PDA from premalignancy to metastatic disease, we found that AnxA2 promoted metastases in vivo. The expression of AnxA2 promoted the secretion of Sema3D from PDA cells, which coimmunoprecipitated with the co-receptor plexin D1 (PlxnD1) on PDA cells. Mouse PDA cells in which SEMA3D was knocked down or ANXA2-null PDA cells exhibited decreased invasive and metastatic potential in culture and in mice. However, restoring Sema3D in AnxA2-null cells did not entirely rescue metastatic behavior in culture and in vivo, suggesting that AnxA2 mediates additional prometastatic mechanisms. Patients with primary PDA tumors that have abundant Sema3D have widely metastatic disease and decreased survival compared to patients with tumors that have relatively low Sema3D abundance. Thus, AnxA2 and Sema3D may be new therapeutic targets and prognostic markers of metastatic PDA.

A preclinical murine model of hepatic metastases.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

Development of a novel preclinical pancreatic cancer research model: bioluminescence image-guided focal irradiation and tumor monitoring of orthotopic xenografts.

PURPOSE: We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response. MATERIALS AND METHODS: Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. BLI was correlated to positron emission tomography (PET)/computed tomography (CT) to estimate tumor dimensions. BLI and cone-beam CT (CBCT) were used to compare tumor centroid location and estimate setup error. BLI and CBCT fusion was performed to guide irradiation of tumors using the small animal radiation research platform (SARRP). DNA damage was assessed by γ-H2Ax staining. BLI was used to longitudinally monitor treatment response. RESULTS: Bioluminescence predicted tumor volume (R = 0.8984) and increased linearly as a function of time up to a 10-fold increase in tumor burden. BLI correlated with PET/CT and necropsy specimen in size (P < .05). Two-dimensional BLI centroid accuracy was 3.5 mm relative to CBCT. BLI-guided irradiated pancreatic tumors stained positively for γ-H2Ax, whereas surrounding normal tissues were spared. Longitudinal assessment of irradiated tumors with BLI revealed significant tumor growth delay of 20 days relative to controls. CONCLUSIONS: We have successfully applied the SARRP to a bioluminescent, orthotopic preclinical pancreas cancer model to noninvasively: 1) allow the identification of tumor burden before therapy, 2) facilitate image-guided focal radiation therapy, and 3) allow normalization of tumor burden and longitudinal assessment of treatment response.

Tyrosine 23 phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and metastases of pancreatic cancer.

The aggressiveness of pancreatic ductal adenocarcinoma (PDA) is characterized by its high metastatic potential and lack of effective therapies, which is the result of a lack of understanding of the mechanisms involved in promoting PDA metastases. We identified Annexin A2 (ANXA2), a member of the Annexin family of calcium-dependent phospholipid binding proteins, as a new molecule that promotes PDA invasion and metastases. We found ANXA2 to be a PDA-associated antigen recognized by post-treatment sera of patients who demonstrated prolonged survival following treatment with a PDA-specific vaccine. Cell surface ANXA2 increases with PDA development and progression. Knockdown of ANXA2 expression by RNA interference or blocking with anti-ANXA2 antibodies inhibits in vitro invasion of PDA cells. In addition, post-vaccination patient sera inhibits in vitro invasion of PDA cells, suggesting that therapeutic anti-ANXA2 antibodies are induced by the vaccine. Furthermore, cell-surface localization of ANXA2 is tyrosine 23 phosphorylation-dependent; and tyrosine 23 phosphorylation is required for PDA invasion. We demonstrated that tyrosine 23 phosphorylation resulting in surface expression of ANXA2 is required for TGFß-induced, Rho-mediated epithelial-mesenchymal transition (EMT), linking the cellular function of ANXA2 which was previously shown to be associated with small GTPase-regulated cytoskeletal rearrangements, to the EMT process in PDA. Finally, using mouse PDA models, we showed that shRNA knock-down of ANXA2, a mutation at tyrosine 23, or anti-ANXA2 antibodies, inhibit PDA metastases and prolong mouse survival. Thus, ANXA2 is part of a novel molecular pathway underlying PDA metastases and a new target for development of PDA therapeutics.