Ren-Wang Peng

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.

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.

22P Increased schedule-dependent efficiency of pemetrexed–cisplatin combination therapy eliminates resistant lung cancer stem-like cells associated with EMT

were used. Pharmacological inhibition of HSP90 activity in these cell lines were achieved through geldanamycin and resorcinol derivatives. The response to these inhibitors at different time points was evaluated. Results: Westerns blots indicated that HSP70 and HSP90-a protein expression were increased after 17-AAG, IPI-504, STA9090 and AUY-922 treatments. EGFR, EML4-ALK and CDK4, the oncogenic client proteins studied, were depleted by the HSP90 inhibitors in the NSCLC cell lines. The strong relationship between client driver protein dependence on Hsp90 and the sensitivity to its inhibition was demonstrated in the HCC827 and H3122 cell lines. Conclusions: The reduction of oncogenic client proteins alongside HSP70 and HSP90-a induction could be used as a validated biomarker signature of HSP90 inhibition in the cell lines studied. Future study will be focused on understanding the biological basis for the differential response to these treatments. Legal entity responsible for the study: Instituo de Biomedicina de Sevilla (IBIS) (CSIC, HUVR, Universidad de Sevilla) Funding: Fundación para la Investigación de Sevilla (FISEVI) Disclosure: All authors have declared no conflicts of interest.

23P Prolonged pemetrexed pretreatment increases efficiency of ionizing radiation combination therapy and correlates with the persistence of treatment-induced DNA damage in lung cancer cells.

pools could sensitize cancer cells to subsequent treatment with cisplatin. Methods: NSCLC A549 cells were treated with pemetrexed for 72 hours. In addition, 24 hours of cisplatin treatment was initiated at day 1, 2 or 3 resulting in either simultaneous pemetrexed application or pemetrexed pretreatment for 24 or 48 hours, respectively. Cell growth and colony formation as well as senescence induction were quantified after treatment. Cell cycle distribution and DNA damage induction was quantified by flow cytometry. Results: Extended pemetrexed pretreatment for 48 hours prior to cisplatin treatment maximally delayed long-term cell growth and significantly reduced the number of recovering clones. Moreover, apoptosis and senescence were augmented and recovery from treatment-induced DNA damage was delayed. Interestingly, a resistant cell population was identified that displayed an epithelialto-mesenchymal transition and which had a stem cell phenotype. Conclusions: Prolonged pemetrexed pretreatment optimizes the anticancer efficiency of pemetrexed–cisplatin combination therapy, therefore, this study warrants further investigations to elucidate whether such an adaptation could enhance the effectiveness of the standard clinical treatment regimen. In addition, a subpopulation of therapy resistant cells with EMT and cancer stem cell features was identified. Legal entity responsible for the study: N/A Funding: Supported by the Bernese Cancer League and the Swiss Cancer Research (KFS-3530–08–2014) to TMM Disclosure: All authors have declared no conflicts of interest.

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.