Sean Hall

Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer

Anticancer therapies currently used in the clinic often can neither eradicate the tumor nor prevent disease recurrence due to tumor resistance. In this study, we showed that chemoresistance to pemetrexed, a multi-target anti-folate (MTA) chemotherapeutic agent for non-small cell lung cancer (NSCLC), is associated with a stem cell-like phenotype characterized by an enriched stem cell gene signature, augmented aldehyde dehydrogenase activity and greater clonogenic potential. Mechanistically, chemoresistance to MTA requires activation of epithelial-to-mesenchymal transition (EMT) pathway in that an experimentally induced EMT per se promotes chemoresistance in NSCLC and inhibition of EMT signaling by kaempferol renders the otherwise chemoresistant cancer cells susceptible to MTA. Relevant to the clinical setting, human primary NSCLC cells with an elevated EMT signaling feature a significantly enhanced potential to resist MTA, whereas concomitant administration of kaempferol abrogates MTA chemoresistance, regardless of whether it is due to an intrinsic or induced activation of the EMT pathway. Collectively, our findings reveal that a bona fide activation of EMT pathway is required and sufficient for chemoresistance to MTA and that kaempferol potently regresses this chemotherapy refractory phenotype, highlighting the potential of EMT pathway inhibition to enhance chemotherapeutic response of lung cancer.

Primary Human Lung Pericytes Support and Stabilize In Vitro Perfusable Microvessels.

The formation of blood vessels is a complex tissue-specific process that plays a pivotal role during developmental processes, in wound healing, cancer progression, fibrosis, and other pathologies. To study vasculogenesis and vascular remodeling in the context of the lung, we developed an in vitro microvascular model that closely mimics the human lung microvasculature in terms of three-dimensional architecture, accessibility, functionality, and cell types. Human pericytes from the distal airway were isolated and characterized using flow cytometry. To assess their role in the generation of normal microvessels, lung pericytes were mixed in fibrin gel and seeded into well-defined microcompartments together with primary endothelial cells (human umbilical cord vein endothelial cells). Patent microvessels covering an area of 3.1 mm(2) formed within 3-5 days and were stable for up to 14 days. Soluble signals from the lung pericytes were necessary to establish perfusability, and pericytes migrated toward endothelial microvessels. Cell-cell communication in the form of adherens and tight junctions, as well as secretion of basement membrane were confirmed using transmission electron microscopy and immunocytochemistry on chip. Direct coculture of pericytes with endothelial cells decreased the microvascular permeability by one order of magnitude from 17.8×10(-6) to 2.0×10(-6) cm/s and led to vessels with significantly smaller and less variable diameter. Upon phenylephrine administration, vasoconstriction was observed in microvessels lined with pericytes, but not in endothelial microvessels only. Perfusable microvessels were also generated with human lung microvascular endothelial cells and lung pericytes. Human lung pericytes were thus shown to have a prominent influence on microvascular morphology, permeability, vasoconstriction, and long-term stability in an in vitro microvascular system. This biomimetic platform opens new possibilities to test functions and interactions of patient-derived cells in a physiologically relevant microvascular setting.

Increased PD-L1 expression and IL-6 secretion characterize human lung tumor-derived perivascular-like cells that promote vascular leakage in a perfusable microvasculature model.

Pericytes represent important support cells surrounding microvessels found in solid organs. Emerging evidence points to their involvement in tumor progression and metastasis. Although reported to be present in the human lung, their specific presence and functional orientation within the tumor microenvironment in non-small cell lung cancer (NSCLC) has not yet been adequately studied. Using a multiparameter approach, we prospectively identified, sorted and expanded mesenchymal cells from human primary NSCLC samples based on co-expression of CD73 and CD90 while lacking hematopoietic and endothelial lineage markers (CD45, CD31, CD14 and Gly-A) and the epithelial marker EpCAM. Compared to their normal counterpart, tumor-derived Lineage-EpCAM-CD73+CD90+ cells showed enhanced expression of the immunosuppressive ligand PD-L1, a higher constitutive secretion of IL-6 and increased basal αSMA levels. In an in vitro model of 3D microvessels, both tumor-derived and matched normal Lineage-EpCAM-CD73+CD90+ cells supported the assembly of perfusable vessels. However, tumor-derived Lineage-EpCAM-CD73+CD90+ cells led to the formation of vessels with significantly increased permeability. Together, our data show that perivascular-like cells present in NSCLC retain functional abnormalities in vitro. Perivascular-like cells as an eventual target in NSCLC warrants further investigation.

Adverse prognostic value of PD-L1 expression in primary resected pulmonary squamous cell carcinomas and paired mediastinal lymph node metastases

Immunohistochemical assessment of programmed cell death (PD)-ligand 1 (PD-L1) expression in lung cancer in the context of therapeutically targeting the PD1/PD-L1 axis is still controversially discussed. This includes the comparability of antibody clones, prognostic value, and discrepancies between primary tumors and metastases. We assessed tumoral PD-L1 expression using clones E1L3N and SP142 in 372 primary resected pulmonary squamous cell carcinomas, including 40 paired N2 lymph node metastases, in relation with clinico-pathological parameters. PD-L1 expression was negative (<1%) in 163/372 (44%, E1L3N) or 231/370 patients (62%, SP142). Positivity of 1–<50% was observed in 135 (36%, E1L3N) or 92 patients (25%, SP142) and ≥50% in 74 (20%, E1L3N) or 47 patients (13%, SP142). PD-L1 staining correlated significantly between both antibodies (r=0.781; P<0.001). Scores correlated significantly between full-slide sections (N=40) and tissue microarrays, and between primaries and N2 metastases (P<0.001 all). CD8+ tumor infiltrating lymphocyte counts positively correlated with PD-L1 expression (P<0.001). PD-L1 ≥50% showed the best prognostic discrimination using the split-sample validation method. It was associated with shorter disease-specific survival in the observation group (E1L3N: P=0.035, SP142: P=0.002) and validation group (E1L3N: P=0.024, SP142: P=0.101) and shorter time to recurrence (observation group: E1L3N: P=0.056, SP142: P<0.001; validation group: E1L3N: P=0.036, SP142: P=0.247). Multivariate analysis showed that PD-L1 expression ≥50% determined by clone E1L3N was an independent prognostic factor in the observation group regarding disease-specific survival (HR=2.768; 95% CI=1.149–6.666; P=0.023) and time to recurrence (HR=2.164; 95% CI=1.056–4.436; P=0.035) and in the validation group (disease-specific survival: HR=1.978; 95% CI=0.928–4.214; P=0.077 and time to recurrence: HR=1.571; 95% CI=0.838–2.944; P=0.159). High PD-L1 expression was associated with adverse prognosis in pulmonary squamous cell carcinoma. Clone E1L3N was more sensitive than SP142 and superior regarding prognostication. PD-L1 expression correlated significantly between primary tumor and N2 metastases, rendering mediastinal lymph node metastases adequate for immunohistochemical assessment.

A Triple Co-Culture Model of the Human Respiratory Tract to Study Immune-Modulatory Effects of Liposomes and Virosomes

The respiratory tract with its ease of access, vast surface area and dense network of antigen-presenting cells (APCs) represents an ideal target for immune-modulation. Bio-mimetic nanocarriers such as virosomes may provide immunomodulatory properties to treat diseases such as allergic asthma. In our study we employed a triple co-culture model of epithelial cells, macrophages and dendritic cells to simulate the human airway barrier. The epithelial cell line 16HBE was grown on inserts and supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) for exposure to influenza virosomes and liposomes. Additionally, primary human nasal epithelial cells (PHNEC) and EpCAM+ epithelial progenitor cell mono-cultures were utilized to simulate epithelium from large and smaller airways, respectively. To assess particle uptake and phenotype change, cell cultures were analyzed by flow cytometry and pro-inflammatory cytokine concentrations were measured by ELISA. All cell types internalized virosomes more efficiently than liposomes in both mono- and co-cultures. APCs like MDMs and MDDCs showed the highest uptake capacity. Virosome and liposome treatment caused a moderate degree of activation in MDDCs from mono-cultures and induced an increased cytokine production in co-cultures. In epithelial cells, virosome uptake was increased compared to liposomes in both mono- and co-cultures with EpCAM+ epithelial progenitor cells showing highest uptake capacity. In conclusion, all cell types successfully internalized both nanocarriers with virosomes being taken up by a higher proportion of cells and at a higher rate inducing limited activation of MDDCs. Thus virosomes may represent ideal carrier antigen systems to modulate mucosal immune responses in the respiratory tract without causing excessive inflammatory changes.

18P Epithelial-to-mesenchymal transition (EMT) is required for resistance to anti-folate chemotherapy in lung cancer.

cells. Prognoscan assessment identified decrease SASH1 mRNA expression lead to a prognostic reduction in patient survival. The depletion of SASH1 in lung cells resulted in a significant increase in cellular proliferation in cancer lung cells. Connectivity mapping predicted the drug Chloropyramine would lead to an increase in SASH1 expression. We demonstrated that Chloropyramine upregulates SASH1 in malignant cell lines. In keeping with this we have demonstrated that Chloropyramine inhibited lung cancer proliferation in vitro. These novel observations support the tumour suppressive role of SASH1 in lung tumourgenesis. Further work is ongoing to understand the function of SASH1 in lung cancer growth. Conclusions: The upregulation of SASH1, either by chemical agents or gene therapy, is a potential novel approach to the management of lung cancer and other solid tumours. Legal entity responsible for the study: Queensland University of Technology Funding: Queensland Health Disclosure: All authors have declared no conflicts of interest.

mTOR mediates a mechanism of resistance to chemotherapy and defines a rational combination strategy to treat KRAS-mutant lung cancer.

Oncogenic KRAS mutations comprise the largest subset of lung cancer defined by genetic alterations, but in the clinic no targeted therapies are available that effectively control mutational KRAS activation. Consequently, patients with KRAS-driven tumors are routinely treated with cytotoxic chemotherapy, which is often transiently effective owing to development of drug resistance. In this study, we show that hyperactivated mammalian target of rapamycin (mTOR) pathway is a characteristic hallmark of KRAS-mutant lung adenocarcinoma after chemotherapy treatment, and that KRAS-mutant lung cancer cells rely on persistent mTOR signaling to resist chemotherapeutic drugs. Coherently, mTOR inhibition circumvents the refractory phenotype and restores sensitivity of resistant KRAS-mutant lung cancer cells to chemotherapy. Importantly, drug combinations of clinically approved mTOR inhibitors and chemotherapy drugs synergize in inhibiting cell proliferation of KRAS-mutant cancer cells in vitro and in vivo, and the efficacy of this combination treatment correlates with the magnitude of mTOR activity induced by chemotherapy alone. These results pinpoint mTOR as a mechanism of resistance to chemotherapy in KRAS-mutant lung cancer and validate a rational and readily translatable strategy that combines mTOR inhibitors with standard chemotherapy to treat KRAS-mutant adenocarcinoma, the most common and deadliest lung cancer subset.

Increased sensitivity to apoptosis upon endoplasmic reticulum stress-induced activation of the unfolded protein response in chemotherapy-resistant malignant pleural mesothelioma

BackgroundStandard treatment for advanced malignant pleural mesothelioma (MPM) is a cisplatin/pemetrexed (MTA) regimen; however, this is confronted by drug resistance. Proteotoxic stress in the endoplasmic reticulum (ER) is a hallmark of cancer and some rely on this stress signalling in response to cytotoxic chemotherapeutics. We hypothesise that ER stress and the adaptive unfolded protein response (UPR) play a role in chemotherapy resistance of MPM.MethodsIn vitro three-dimensional (3D) and ex vivo organotypic culture were used to enrich a chemotherapy-resistant population and recapitulate an in vivo MPM microenvironment, respectively. Markers of ER stress, the UPR and apoptosis were assessed at mRNA and protein levels. Cell viability was determined based on acid phosphatase activity.ResultsMPM cells with de novo and/or acquired chemotherapy resistance displayed low ER stress, which rendered the cells hypersensitive to agents that induce ER stress and alter the UPR. Bortezomib, an FDA-approved proteasome inhibitor, selectively impairs chemotherapy-resistant MPM cells by activating the PERK/eIF2α/ATF4-mediated UPR and augmenting apoptosis.ConclusionsWe provide the first evidence for ER stress and the adaptive UPR signalling in chemotherapy resistance of MPM, which suggests that perturbation of the UPR by altering ER stress is a novel strategy to treat chemotherapy-refractory MPM.

Virosome-bound antigen enhances DC-dependent specific CD4+ T cell stimulation, inducing a Th1 and Treg profile in vitro

There is considerable interest to develop antigen-carriers for immune-modulatory clinical applications, but insufficient information is available on their effects on antigen-presenting cells. We employed virosomes coupled to ovalbumin (OVA) to study their interaction with murine bone marrow-derived dendritic cells (BMDCs) and modulation of downstream T cell responses. BMDCs were treated in vitro with virosomes or liposomes prior to determining BMDC phenotype, viability, and intracellular trafficking. Antigen-specific CD4+ T cell activation was measured by co-culture of BMDCs with DO11.10 CD4+ T cells. Compared to liposomes, virosomes were rapidly taken up. Neither nanocarrier type affected BMDC viability, nor did a moderate degree of activation differ for markers such as CD40, CD80, CD86. Virosome uptake occurred via clathrin-mediated endocytosis and phagocytosis, with co-localization in late endosomes. Only BMDCs treated with OVA-coupled virosomes induced enhanced OVA-specific CD4+ T cell proliferation. Antigen-coupled virosomes are endowed with an intrinsic ability to modulate DC-dependent adaptive immune responses.

10PDAssessment of PD-L1 and CD47 expression together with tumor-associated TILs in resectable early stage NSCLC

Aim/Background: Solid tumors have been shown to evade host antitumor immunity through upregulation of the immune checkpoint PD-1/PD-L1 pathway. However, CD47, an antiphagocytic ligand expressed on tumor cells, represents another cell surface molecule promoting tumor immune evasion via targeting the innate immune system. We investigated whether PD-L1 and CD47 are co-expressed in early stage non-small cell lung cancer (NSCLC). Methods: Resected tumor and matched adjacent normal tissue from 84 stage I-III NSCLCs (42 adenocarcinomas [Adeno]; 42 squamous cell carcinomas [SqCC]) were processed to single cell suspensions, stained with a panel of antibodies (CD45, CD31, CD14, EpCAM, CD73, CD90, PD-L1, CD47) and subjected to multicolor flow cytometric analysis. In parallel, the phenotype of tumor infiltrating lymphocytes (TILs) was also performed using CD19, CD45RO, CD3, CD4, CD8, PD-1, CD127 and CD107a. In a subset of patients, expression of PD-L1 and CD47 was confirmed via immunohistochemistry. Results: PD-L1 expression on tumor epithelium (EpCAM+) was increased in both Adeno (p = 0.0295) and SqCC (p = 0.0016) patients. CD47 was also upregulated on the tumor EpCAM+ fraction in SqCC (p = 0.05) but not in Adeno patients (p = 0.124). An increase in the tumor mesenchymal fraction in both Adeno (p = 0.015) and SqCC (p = 0.027) patients was found that showed an enhanced expression of PD-L1 but not CD47. Immunohistochemical analysis confirmed coexpression of PD-L1 and CD47 in a subset of patients. Finally, there was an increase in CD4+PD-1hi TILs in Adeno patients, whereas a decrease in CD127 expression was found on CD8+ TILs (p < 0.0001) with a CD107aloPD1hi phenotype in both histological subtypes. Conclusions: We detected PD-L1 expression in different compartments in a subset of patients with early stage Adeno and SqCC, whereas CD47 expression was restricted to the tumor epithelial compartment in SqCC patients only. These differences may be related to the intragraft immune priming inside the tumor microenvironment. Further study is required to determine whether dual targeting of PD-L1 and CD47 in the perioperative setting represents a promising therapeutic strategy to reinvigorate TILs in affected patients. Legal entity responsible for the study: N/A Funding: Inselspital, University Hospital of Bern, Division of Thoracic Surgery Disclosure: All authors have declared no conflicts of interest.