Immunomodulatory Effects of Stereotactic Body Radiotherapy and Vaccination with Heat-Killed Mycobacterium Obuense (IMM-101) in Patients with Locally Advanced Pancreatic Cancer.

BACKGROUND: Patients with locally advanced pancreatic cancer (LAPC) are treated with chemotherapy. In selected cases, stereotactic body radiotherapy (SBRT) can be added to the regimen. We hypothesized that adding an adjuvant containing a heat-killed mycobacterium (IMM-101) to SBRT may lead to beneficial immuno-modulatory effects, thereby improving survival. This study aims to investigate the safety of adding IMM-101 to SBRT and to investigate the immuno-modulatory effects of the combination treatment in the peripheral blood of LAPC patients. METHODS: LAPC patients were treated with SBRT (40 Gy) and six intradermal vaccinations of one milligram IMM-101. The primary endpoint was an observed toxicity rate of grade 4 or higher. Targeted gene-expression profiling and multicolor flow cytometry were performed for longitudinal immune-monitoring of the peripheral blood. RESULTS: Twenty patients received study treatment. No treatment-related adverse events of grade 4 or higher occurred. SBRT/IMM-101 treatment induced a transient decrease in different lymphocyte subsets and an increase in CD14+CD16-CD11b+HLA-DRlow myeloid-derived suppressor cells. Importantly, treatment significantly increased activated ICOS+, HLA-DR+ and Ki67+PD1+ T and NK cell frequencies. This was not accompanied by increased levels of most inhibitory markers, such as TIM-3 and LAG-3. CONCLUSIONS: Combination therapy with SBRT and a heat-killed mycobacterium vaccine was safe and had an immune-stimulatory effect.

Immunomodulatory antitumor effect of interferon­beta combined with gemcitabine in pancreatic cancer.

Resistance to gemcitabine is common and critically limits its therapeutic efficacy in patients with pancreatic cancer. Interferon­beta (IFN­ß) induces numerous antitumor effects and synergizes with gemcitabine treatment. The immunomodulatory effects of this treatment regimen have not yet been described. In the present study, the antitumor effect of IFN­ß combined with gemcitabine was investigated in immune competent mice. Mouse KPC3 cells were used in all experiments. Treatment effects were determined with cell proliferation assay. Reverse transcription­quantitative PCR was used to measure gene expression. For in vivo experiments, cells were subcutaneously injected in immune competent mice. For immune profiling, NanoString analysis was performed on tumor samples of treated and untreated mice. Baseline expression of Ifnar­1 and Ifnar­2c in KPC3 cells was 1.42±0.16 and 1.50±0.17, respectively. IC50 value of IFN­ß on cell growth was high (>1,000 IU/ml). IFN­ß pre­treatment increased the in vitro response to gemcitabine (1.3­fold decrease in EC50; P<0.001). In vivo, tumor size was not statistically significant smaller in mice treated with IFN­ß plus gemcitabine (707±92 mm3 vs. 1,239±338 mm3 in vehicle­treated mice; P=0.16). IFN­ß alone upregulated expression of numerous immune­related genes. This effect was less pronounced when combined with gemcitabine. For the first time, to the best of our knowledge, the immunomodulatory effects of IFN­ß, alone and combined with gemcitabine, in pancreatic cancer were reported. Prognostic markers for predicting effective responses to IFN­ß therapy are urgently needed.

The Class I HDAC Inhibitor Valproic Acid Strongly Potentiates Gemcitabine Efficacy in Pancreatic Cancer by Immune System Activation.

Background: Gemcitabine efficacy in pancreatic cancer is often impaired due to limited intracellular uptake and metabolic activation. Epi-drugs target gene expression patterns and represent a promising approach to reverse chemoresistance. In this study, we investigate the chemosensitizing effect of different epi-drugs when combined with gemcitabine in pancreatic cancer. Methods: Mouse KPC3 cells were used for all experiments. Five different epi-drugs were selected for combination therapy: 5-aza-2'-deoxycytidine, hydralazine, mocetinostat, panobinostat, and valproic acid (VPA). Treatment effects were determined by cell proliferation and colony forming assays. Expression of genes were assessed by real-time quantitative PCR. The most promising epi-drug for combination therapy was studied in immune competent mice. Intratumor changes were defined using NanoString PanCancer panel IO360. Results: All epi-drugs, except hydralazine, potentiated the gemcitabine response in KPC3 cells (range decrease IC50 value 1.7−2-fold; p < 0.001). On colony formation, the cytotoxic effect of 0.5 ng/mL gemcitabine was 1.4 to 6.3 times stronger (p < 0.01). Two out of three drug-transporter genes were strongly upregulated following epi-drug treatment (a range fold increase of 17−124 and 9−60 for Slc28a1 and Slc28a3, respectively; all p < 0.001). VPA combined with gemcitabine significantly reduced tumor size with 74% compared to vehicle-treated mice and upregulated expression of immune-related pathways (range pathway score 0.86−1.3). Conclusions: These results provide a strong rationale for combining gemcitabine with VPA treatment. For the first time, we present intratumor changes and show activation of the immune system. Clinical trials are warranted to assess efficacy and safety of this novel combination in pancreatic cancer patients.

The Placental Innate Immune System Is Altered in Early-Onset Preeclampsia, but Not in Late-Onset Preeclampsia.

Preeclampsia is a severe placenta-related pregnancy disorder that is generally divided into two subtypes named early-onset preeclampsia (onset <34 weeks of gestation), and late-onset preeclampsia (onset ≥34 weeks of gestation), with distinct pathophysiological origins. Both forms of preeclampsia have been associated with maternal systemic inflammation. However, alterations in the placental immune system have been less well characterized. Here, we studied immunological alterations in early- and late-onset preeclampsia placentas using a targeted expression profile approach. RNA was extracted from snap-frozen placenta samples (healthy n=13, early-onset preeclampsia n=13, and late-onset preeclampsia n=6). The expression of 730 immune-related genes from the Pan Cancer Immune Profiling Panel was measured, and the data were analyzed in the advanced analysis module of nSolver software (NanoString Technology). The results showed that early-onset preeclampsia placentas displayed reduced expression of complement, and toll-like receptor (TLR) associated genes, specifically TLR1 and TLR4. Mast cells and M2 macrophages were also decreased in early-onset preeclampsia compared to healthy placentas. The findings were confirmed by an immunohistochemistry approach using 20 healthy, 19 early-onset preeclampsia, and 10 late-onset preeclampsia placentas. We conclude that the placental innate immune system is altered in early-onset preeclampsia compared to uncomplicated pregnancies. The absence of these alterations in late-onset preeclampsia placentas indicates dissimilar immunological profiles. The study revealed distinct pathophysiological processes in early-onset and late-onset preeclampsia placentas and imply that a tailored treatment to each subtype is desirable.

Periostin Is Expressed by Pericytes and Is Crucial for Angiogenesis in Glioma.

The expression of the matricellular protein periostin has been associated with glioma progression. In previous work we found an association of periostin with glioma angiogenesis. Here, we screen gliomas for POSTN expression and identify the cells that express periostin in human gliomas. In addition, we study the role of periostin in an in vitro model for angiogenesis. The expression of periostin was investigated by RT-PCR and by immunohistochemistry. In addition, we used double labeling and in situ RNA techniques to identify the expressing cells. To investigate the function of periostin, we silenced POSTN in a 3D in vitro angiogenesis model. Periostin expression was elevated in pilocytic astrocytoma and glioblastoma, but not in grade II/III astrocytomas and oligodendrogliomas. The expression of periostin colocalized with PDGFRß+ cells, but not with OLIG2+/SOX2+ glioma stem cells. Silencing of periostin in pericytes in coculture experiments resulted in attenuation of the numbers and the length of the vessels formation and in a decrease in endothelial junction formation. We conclude that pericytes are the main source of periostin in human gliomas and that periostin plays an essential role in the growth and branching of blood vessels. Therefore, periostin should be explored as a novel target for developing anti-angiogenic therapy for glioma.

Dendritic cell vaccination and CD40-agonist combination therapy licenses T cell-dependent antitumor immunity in a pancreatic carcinoma murine model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. METHODS: Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. RESULTS: Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. CONCLUSION: These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.