PD-L1 Dependent Immunogenic Landscape in Hot Lung Adenocarcinomas Identified by Transcriptome Analysis

Background: Lung cancer is the most frequent cause of cancer-related deaths worldwide. The clinical development of immune checkpoint blockade has dramatically changed the treatment paradigm for patients with lung cancer. Yet, an improved understanding of PD-1/PD-L1 checkpoint blockade- responsive biology is warranted. Methods: We aimed to identify the landscape of immune cell infiltration in primary lung adenocarcinoma (LUAD) in the context of tumoral PD-L1 expression and the extent of immune infiltration ("hot" vs. "cold" phenotype). The study comprises LUAD cases (n = 138) with "hot" (≥150 lymphocytes/HPF) and "cold" (<150 lymphocytes/HPF) tumor immune phenotype and positive (>50%) and negative (<1%) tumor PD-L1 expression, respectively. Tumor samples were immunohistochemically analyzed for expression of PD-L1, CD4, and CD8, and further investigated by transcriptome analysis. Results: Gene set enrichment analysis defined complement, IL-JAK-STAT signaling, KRAS signaling, inflammatory response, TNF-alpha signaling, interferon-gamma response, interferon-alpha response, and allograft rejection as significantly upregulated pathways in the PD-L1-positive hot subgroup. Additionally, we demonstrated that STAT1 is upregulated in the PD-L1-positive hot subgroup and KIT in the PD-L1-negative hot subgroup. Conclusion: The presented study illustrates novel aspects of PD-L1 regulation, with potential biological relevance, as well as relevance for immunotherapy response stratification

Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.

Histological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion of L. pneumophila to the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA(-) strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of L. pneumophila infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations

Novel frontiers in detecting cancer metastasis.

Cancer microenvironment is the critical battle ground between the cancer cells and host response. Thus, more emphasis is directed to study the relationship between cancer cells and the stromal cells. Multiplex microscopy is an emerging technique in which multiple cell populations within the cancer microenvironment may be stained so that spatial relationship between cancer cells and, in particular, the immune cells may be studied during different stages of cancer development. Recent discovery of mutational burden and neoantigens in cancer has opened new landscapes in the interaction of host immune cells and cancer neoantigens. The emerging role of miRNAs may become an added dimension to study cancer beyond traditional pathway of DNA directed RNA being associated with the malignant behavior of cancer. Circulating tumor cells, cancer markers and ctDNA can be used as markers for circulating cancer cells in the blood. Further studies are needed to validate if liquid biopsy of cancer may become a routine clinical tool to screen cancer or follow patients for recurrence or responses to treatment

The human placenta releases substances that drive lung cancer into apoptosis

BACKGROUND: As there is no optimal treatment of non small cell lung cancer due to its resistance to common chemotherapeutics, we investigated the effect of human placenta-conditioned medium on tumor tissue. The human placenta constitutes a mixture of maternal and fetal origin and displays a variety of immunomodulatory aspects. METHODS: Freshly resected non small cell lung cancer tissues were incubated with placenta-conditioned medium in a short-term tissue culture model and A549 cells were challenged, respectively. Term placenta was used for producing conditioned medium and HOPE-fixed stimulated tumor tissue was analyzed for expression of caspase-3 and Ki67 via immunohistochemistry. The effects of conditioned medium on squamous cell carcinoma were further compared to physiological concentrations of Carboplat/Gemzar. RESULTS: Conditioned medium caused in 2 of 3 cases elevated expression of caspase-3 and reduced expression of Ki67 in 3 out of 3 cases, while the chemotherapeutic agents caused no comparable expression of caspase-3 or reduction of Ki67. In cell culture up to 50% of karyopyknosis was investigated and even sterile-filtrated medium caused widespread reduction of Ki67 on protein level. CONCLUSION: Human placenta releases substances that mediate apoptosis and reduce proliferation in tumor tissue and cell culture. As even sterile-filtrated medium caused the mentioned effects we hypothesize one or more soluble mediators. The detailed way of promoting apoptosis and nature of these mediators need to be elucidated in further studies

The human placenta releases substances that drive lung cancer into apoptosis

Abstract Background As there is no optimal treatment of non small cell lung cancer due to its resistance to common chemotherapeutics, we investigated the effect of human placenta-conditioned medium on tumor tissue. The human placenta constitutes a mixture of maternal and fetal origin and displays a variety of immunomodulatory aspects. Methods Freshly resected non small cell lung cancer tissues were incubated with placenta-conditioned medium in a short-term tissue culture model and A549 cells were challenged, respectively. Term placenta was used for producing conditioned medium and HOPE-fixed stimulated tumor tissue was analyzed for expression of caspase-3 and Ki67 via immunohistochemistry. The effects of conditioned medium on squamous cell carcinoma were further compared to physiological concentrations of Carboplat/Gemzar. Results Conditioned medium caused in 2 of 3 cases elevated expression of caspase-3 and reduced expression of Ki67 in 3 out of 3 cases, while the chemotherapeutic agents caused no comparable expression of caspase-3 or reduction of Ki67. In cell culture up to 50% of karyopyknosis was investigated and even sterile-filtrated medium caused widespread reduction of Ki67 on protein level. Conclusion Human placenta releases substances that mediate apoptosis and reduce proliferation in tumor tissue and cell culture. As even sterile-filtrated medium caused the mentioned effects we hypothesize one or more soluble mediators. The detailed way of promoting apoptosis and nature of these mediators need to be elucidated in further studies.</p

Phosphorylation of SMAD3 in immune cells predicts survival of patients with early stage non-small cell lung cancer.

BACKGROUND: The interplay of immune and cancer cells takes place in the tumor microenvironment where multiple signals are exchanged. The transforming growth factor beta (TGFB) pathway is known to be dysregulated in lung cancer and can impede an effective immune response. However, the exact mechanisms are yet to be determined. Especially which cells respond and where does this signaling take place with respect to the local microenvironment. METHODS: Human non-small cell lung cancer samples were retrospectively analyzed by multiplexed immunohistochemistry for SMAD3 phosphorylation and programmed death ligand 1 expression in different immune cells with respect to their localization within the tumor tissue. Spatial relationships were studied to examine possible cell-cell interactions and analyzed in conjunction with clinical data. RESULTS: TGFB pathway activation in CD3, CD8, Foxp3 and CD68 cells, as indicated by SMAD3 phosphorylation, negatively impacts overall and partially disease-free survival of patients with lung cancerindependent of histological subtype. A high frequency of Foxp3 regulatory T cells positive for SMAD3 phosphorylation in close vicinity of CD8 T cells within the tumor discriminate a rapidly progressing group of patients with lung cancer. CONCLUSIONS: TGFB pathway activation of local immune cells within the tumor microenvironment impacts survival of early stage lung cancer. This might benefit patients not eligible for targeted therapies or immune checkpoint therapy as a therapeutic option to re-activate the local immune response

Neutrophil extracellular trap formation and extracellular DNA in sputum of stable COPD patients

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic airway inflammation. Neutrophil extracellular trap (NET) formation - a meshwork of neutrophil DNA components and neutrophil enzymes are involved in innate immunity and inflammation. Little is known about the presence of these structures in induced sputum from stable COPD patients. METHODS: Induced sputum samples of 23 COPD patients and 10 healthy controls were collected. Sputum cells were harvested, cultivated and stained for NET components. Extracellular DNA was quantified using a NanoDrop 2000 spectrophotometer. RESULTS: NET formation was markedly upregulated in COPD sputum compared with healthy controls, irrespective of sputum purulence or smoking status. NET formation was associated with significantly higher concentration of extracellular DNA in sputum supernatant (484 ng/mul in COPD versus 268 ng/mul in controls, p = 0.013). Log-transformed extracellular DNA correlated with log-transformed absolute neutrophil numbers in sputum (r = 0.60; p < 0.001) and airway obstruction (r = -0.43; p = 0.013). CONCLUSION: NET formation associated with higher concentrations of extracellular DNA may be a pathobiological feature of COPD-derived sputum neutrophils

Future Research Goals in Immunotherapy.

In our opinion the most urgent needs to improve patient outcomes are: 1) a deeper ability to measure cancer immunobiology, and 2) increased availability of agents that, coupled with predictive biomarkers, will be used to tailor anti-cancer immunity. Tailoring effective immunotherapy will entail combinations of immunotherapeutics that augment priming of anti-cancer immunity, boost expansion of effector and memory cells of the T, B and NK lineage, amplify innate immunity and relieve checkpoint inhibition. Alternatives to inducing adaptive immunity to cancer include synthetic immunology that incorporate bi-specifics that target T cells to cancer or adoptive immunotherapy with gene-modified immune cells