Long Non-Coding RNA THOR Depletion Inhibits Human Non-Small Cell Lung Cancer Cell Growth

Long non-coding RNA (LncRNA) THOR (Lnc-THOR) is expressed in testis and multiple human malignancies. Lnc-THOR association with IGF2BP1 (IGF2 mRNA-binding protein 1) is essential for stabilization and transcription of IGF2BP1 targeted mRNAs. We tested its expression and potential functions in non-small cell lung cancer (NSCLC). In primary NSCLC cells and established cell lines, Lnc-THOR shRNA or CRISPR/Cas9-mediated knockout (KO) downregulated IGF2BP1 target mRNAs (IGF2, Gli1, Myc and SOX9), inhibiting cell viability, growth, proliferation, migration and invasion. Significant apoptosis activation was detected in Lnc-THOR-silenced/-KO NSCLC cells. Conversely, ectopic overexpression of Lnc-THOR upregulated IGF2BP1 mRNA targets and enhanced NSCLC cell proliferation, migration and invasion. RNA-immunoprecipitation and RNA pull-down assay results confirmed the direct binding between Lnc-THOR and IGF2BP1 protein in NSCLC cells. Lnc-THOR silencing and overexpression were ineffective in IGF2BP1-KO NSCLC cells. Forced IGF2BP1 overexpression failed to rescue Lnc-THOR-KO NSCLC cells. In vivo, intratumoral injection of Lnc-THOR shRNA adeno-associated virus potently inhibited A549 xenograft tumor growth in nude mice. At last we show that Lnc-THOR is overexpressed in multiple NSCLC tissues and established/primary NSCLC cells. Collectively, these results highlighted the ability of Lnc-THOR in promoting NSCLC cell growth by associating with IGF2BP1, suggesting that Lnc-THOR represents a promising therapeutic target of NSCLC.

Synergistic cytotoxicity of the CDK4 inhibitor Fascaplysin in combination with EGFR inhibitor Afatinib against Non-small Cell Lung Cancer

SummaryIn the absence of suitable molecular markers, non-small cell lung cancer (NSCLC) patients have to be treated with chemotherapy with poor results at advanced stages. Therefore, the activity of the anticancer marine drug fascaplysin was tested against primary NSCLC cell lines established from pleural effusions. Cytotoxicity of the drug or combinations were determined using MTT assays and changes in intracellular phosphorylation by Western blot arrays. Fascaplysin revealed high cytotoxicity against NSCLC cells and exhibit an activity pattern different of the standard drug cisplatin. Furthermore, fascaplysin synergizes with the EGFR tyrosine kinase inhibitor (TKI) afatinib to yield a twofold increased antitumor effect. Interaction with the Chk1/2 inhibitor AZD7762 confirm the differential effects of fascplysin and cisplatin. Protein phosphorylation assays showed hypophosphorylation of Akt1/2/3 and ERK1/2 as well as hyperphosphorylation of stress response mediators of H1299 NSCLC cells. In conclusion, fascaplysin shows high cytotoxicity against pleural primary NSCLC lines that could be further boosted when combined with the EGFR TKI afatinib.

Innate immune activation by checkpoint inhibition in human patient-derived lung cancer tissues

Although Pembrolizumab-based immunotherapy has significantly improved lung cancer patient survival, many patients show variable efficacy and resistance development. A better understanding of the drug's action is needed to improve patient outcomes. Functional heterogeneity of the tumor microenvironment (TME) is crucial to modulating drug resistance; understanding of individual patients' TME that impacts drug response is hampered by lack of appropriate models. Lung organotypic tissue slice cultures (OTC) with patients' native TME procured from primary and brain-metastasized (BM) non-small cell lung cancer (NSCLC) patients were treated with Pembrolizumab and/or beta-glucan (WGP, an innate immune activator). Metabolic tracing with 13C6-Glc/13C5,15N2-Gln, multiplex immunofluorescence (mIF), and digital spatial profiling (DSP) were employed to interrogate metabolic and functional responses to Pembrolizumab and/or WGP. Primary and BM PD-1+ lung cancer OTC responded to Pembrolizumab and Pembrolizumab + WGP treatments, respectively. Pembrolizumab activated innate immune metabolism and functions in primary OTC, which were accompanied by tissue damage. DSP analysis indicated an overall decrease in immunosuppressive macrophages and T cells but revealed microheterogeneity in immune responses and tissue damage. Two TMEs with altered cancer cell properties showed resistance. Pembrolizumab or WGP alone had negligible effects on BM-lung cancer OTC but Pembrolizumab + WGP blocked central metabolism with increased pro-inflammatory effector release and tissue damage. In depth metabolic analysis and multiplex TME imaging of lung cancer OTC demonstrated overall innate immune activation by Pembrolizumab but heterogeneous responses in the native TME of a patient with primary NSCLC. Metabolic and functional analysis also revealed synergistic action of Pembrolizumab and WGP in OTC of metastatic NSCLC.

SURG-05. Neurosurgery for brain metastases from non-small cell lung cancer: survival outcome and prognostic factors

Background Surgery is an important approach to treat non-small cell lung cancer (NSCLC) brain metastases (BM). Here, we analyzed the survival outcome and prognostic factors for patients with NSCLC after BM resection. Methods The Surveillance Epidemiology and End Results (SEER) database was employed to address the incidence of BM from NSCLC and the current prognosis at population level. 674 contemporaneous NSCLC patients received BM resection at Sun Yat-sen University Cancer Center (SYSUCC) were used for survival comparison and Cox proportional hazards model was applied for identifying prognostic factors. Results 60,436 NSCLC patients diagnosed between 2010 to 2017 were enrolled from SEER database. Among them, 8,708 (14.4%) BM were identified at primary NSCLC diagnosis (synchronous BM, SBM). Median overall survival (OS) of SBM was 6 months with 1-, and 3-year survival percentages of 30.3% and 9.8%, respectively. Furthermore, the survival of BM patients without extracranial metastasis is significantly longer than those with extracranial metastases (median OS: 10 versus 5 months, P<0.001). 225 SBM (cohort A) and 449 BM with treatment history on primary NSCLC (cohort B) were collected from SYSUCC. In cohort A, 86 BM with extracranial metastases were found (38.2%) and the median OS was significantly shorter than those without extracranial metastases (15.2 versus 23.7 months, P<0.001). In cohort B, 255 cases with extracranial metastases were found (56.8%) and their prognosis was also worse than cases without extracranial metastases (median OS: 18.3 versus 22.1 months, P=0.002). Multivariate analyses revealed that younger age (HR=0.71, P=0.003), without extracranial metastases (HR=0.65, P<0.001) and radiation for BM (HR=0.78, P=0.005) were independent factors for better OS. Conclusion Improved survival of patients received BM resection was observed in SYSUCC cohort as comparison with SEER patients with NSCLC and BM. Aggressive local treatment including surgery and radiation is still important in Modern management of BM from NSCLC.

The Anti-Non-Small Cell Lung Cancer Cell Activity by a mTOR Kinase Inhibitor PQR620

In non-small-cell lung carcinoma (NSCLC), aberrant activation of mammalian target of rapamycin (mTOR) contributes to tumorigenesis and cancer progression. PQR620 is a novel and highly-potent mTOR kinase inhibitor. We here tested its potential activity in NSCLC cells. In primary human NSCLC cells and established cell lines (A549 and NCI-H1944), PQR620 inhibited cell growth, proliferation, and cell cycle progression, as well as cell migration and invasion, while inducing significant apoptosis activation. PQR620 disrupted assembles of mTOR complex 1 (mTOR-Raptor) and mTOR complex 2 (mTOR-Rictor-Sin1), and blocked Akt, S6K1, and S6 phosphorylations in NSCLC cells. Restoring Akt-mTOR activation by a constitutively-active Akt1 (S473D) only partially inhibited PQR620-induced cytotoxicity in NSCLC cells. PQR620 was yet cytotoxic in Akt1/2-silenced NSCLC cells, supporting the existence of Akt-mTOR-independent mechanisms. Indeed, PQR620 induced sphingosine kinase 1 (SphK1) inhibition, ceramide production and oxidative stress in primary NSCLC cells. In vivo studies demonstrated that daily oral administration of a single dose of PQR620 potently inhibited primary NSCLC xenograft growth in severe combined immune deficient mice. In PQR620-treated xenograft tissues, Akt-mTOR inactivation, apoptosis induction, SphK1 inhibition and oxidative stress were detected. In conclusion, PQR620 exerted potent anti-NSCLC cell activity via mTOR-dependent and -independent mechanisms.

Untargeted lipidomics of non-small cell lung carcinoma shows differentially abundant lipid classes in cancer vs non-cancer tissue

Lung cancer is the leading cause of cancer death worldwide and non-small cell lung carcinoma (NSCLC) represents 85% of newly diagnosed lung cancers. The high mortality rate of lung cancer is due in part to the lack of effective treatment options for advanced disease. A major limitation in the development of effective treatment options is our incomplete understanding of NSCLC metabolism at a molecular level, especially lipids. Improvements in mass spectrometry combined with our untargeted assignment tool SMIRFE enable the systematic and less biased examination of NSCLC lipid metabolism. Lipids were extracted from paired tumorous and non-tumorous lung tissue samples from 86 patients with suspected resectable stage I or IIa primary NSCLC and were analyzed using ultra-high resolution Fourier transform mass spectrometry. Pathological examination of the samples revealed that the majority of the samples were primary NSCLC; however, the disease group does include examples of metastases of other cancers and several granulomas. Information-content-informed (ICI) Kendall tau correlation analysis revealed correlation and co-occurrence patterns consistent with significant changes in lipid profiles between disease and non-disease samples. Lipids assigned using the program SMIRFE (Mitchell et al., 2019) were analyzed for differential abundance, followed by machine learning to classify the SMIRFE formula assignments into lipid categories. At the lipid category level, sterol abundances were consistently higher in diseased versus non-diseased lung tissues at statistically significant levels. The statistically significant increase in sterol abundances in primary NSCLC compared with non-cancerous lung tissue suggests for treatment of primary NSCLC a possible therapeutic role for statins and nitrogenous bisphosphonates, pharmaceuticals that inhibit endogenous sterol biosynthesis. This hypothesis is consistent with previous epidemiological studies that have identified a therapeutic role for statins in the treatment of NSCLC but were unable to identify a molecular mechanism for this effect. Additionally, the majority of the consistently increased sterol abundances belong to the sterol ester subcategory, suggesting increased SCD1 and ACAT1 activity. SCD1 expression is a known negative prognostic indicator for survival in NSCLC. In our study, a large fraction of the NSCLC samples displayed this phenotype; however, SCD1 mutants would be unexpected in all of these samples. This suggests that this metabolic phenotype may be shared across multiple genetic subtypes of NSCLC. Thus, inhibitors of SCD1 and other enzymes involved in the production of this metabolic phenotype could have utility in the treatment of many genetic subtypes of NSCLC.

Expression of inflammatory interleukins and selected miRNAs in non-small cell lung cancer

Tumours are characterised by an ability to avoid immune destruction and the presence of cancer-associated inflammation. Better understanding of the link between lung cancer and such inflammation is vital for early detection and personalized treatment. Thus, we examined the mRNA expression of interleukins IL-1β, IL-6, IL-17 and miR-9, miR-122 as potential useful biomarkers of NSCLC. Tumour tissues, non-cancerous tissue and blood samples were collected from 39 patients with primary NSCLC undergoing surgical treatment. The selected RNA was isolated from tissue samples and selected miRNAs from peripheral blood exosomes. This RNA was transcribed to cDNA and quantified using RT-qPCR. Significantly higher expression of the selected interleukins was observed in non-cancerous than tumour tissue, and IL-6 was significantly higher in the tumour tissue of patients with a history of ≤ 40 pack-years (PYs) (2.197, IQR: 0.821–4.415) than in those with > 40 PYs (0.461, IQR: 0.372–0.741; p = 0.037). It is clear that inflammatory processes play a role in NSCLC, as indicated by the upregulation of IL-1β and IL-6 in tumour and adjacent tissue, and that smoking has a strong influence on inflammation in tumourigenesis, demonstrated by the upregulation of IL-6 in tumour samples among patients with ≤ 40 PYs compared to > 40 PYs.

Contribution of resident and circulating precursors to tumor-infiltrating CD8+ T cell populations in lung cancer

Tumor-infiltrating lymphocytes (TILs), in general, and especially CD8+ TILs, represent a favorable prognostic factor in non–small cell lung cancer (NSCLC). The tissue origin, regenerative capacities, and differentiation pathways of TIL subpopulations remain poorly understood. Using a combination of single-cell RNA and T cell receptor (TCR) sequencing, we investigate the functional organization of TIL populations in primary NSCLC. We identify two CD8+ TIL subpopulations expressing memory-like gene modules: one is also present in blood (circulating precursors) and the other one in juxtatumor tissue (tissue-resident precursors). In tumors, these two precursor populations converge through a unique transitional state into terminally differentiated cells, often referred to as dysfunctional or exhausted. Differentiation is associated with TCR expansion, and transition from precursor to late-differentiated states correlates with intratumor T cell cycling. These results provide a coherent working model for TIL origin, ontogeny, and functional organization in primary NSCLC.

New aptamer-based approaches for the targeting of cancer associated fibroblast in NSCLC

The aim of the research. Non-small cell lung cancer (NSCLC) represents about 80 % of all lung cancer cases and is oft en associated with drug resistance, relapses and a poor prognosis. Th erefore, the identifi cation of eff ective therapeutic strategies represents a crucial challenge in oncology. A key limit of conventional anticancer treatments is that they do not target the permissive tumor microenvironment, of which key components are cancer‐associated fibroblasts (CAFs). It has been shown that CAFs are able to regulate malignant progression and drug resistance. However, a detailed characterization of CAF profile and the targeting of their pro-tumor eff ects still remain an ambitious challenge and have a primary importance for the identifi cation of new eff ective therapies. Material and methods. We aimed to develop innovative strategies based on nucleic acid aptamers to address these fundamental issues. Firstly, we applied an aptamer conjugate (named Gint4.T-STAT3), containing a STAT3 siRNA linked to an aptamer binding and inhibiting the PDGFRβ, to specifi cally silence STAT3 reported as a fundamental player in the cross-talk between CAFs and epithelial NSCLC cells. Results. We demonstrated that this molecule eff ectively delivers STAT3 siRNA in NSCLC cells, blocking CAF-induced cell growth and migration in both continous and primary NSCLC coltures. In addition, in order to address CAF specifi c targeting and profi ling, we developed an innovative diff erential cell-SELEX approach by using primary NSCLC CAFs as selection target. Such a strategy allowed the isolation of diff erent aptamers discriminating NSCLC CAFs from normal lung fi broblasts. Th e analyses of aptamer specifi city and functionality is curently ongoing. Conclusion. Our data represent the fi rst ever attempt in CAF targeting using aptamer-based drugs, and can open innovative horizons in the current therapeutic approaches forNSCLC.

Contribution of resident and circulating precursors to tumor-infiltrating CD8+ T cell populations in non-small cell lung cancer patients

Tumor-infiltrating lymphocytes (TILs) in general, and CD8+ TILs in particular, represent a favorable prognostic factor in non-small cell lung cancer (NSCLC). The tissue origin, regenerative capacities, and differentiation pathways of TIL subpopulations, however, remain poorly understood. Using a combination of single cell RNA and T cell receptor (TCR) sequencing, we investigate the functional organization of TIL populations in primary NSCLC. We identify two CD8+ TIL subpopulations expressing memory-like gene modules: one is also present in blood (circulating precursors) and the other one in juxta-tumor tissue (tissue resident precursors). In tumors, these two precursor populations converge through a unique transitional state into terminally differentiated cells, often referred to as dysfunctional or exhausted. Differentiation is associated with TCR expansion, and transition from precursor to late differentiated states correlates with intratumor T cell cycling. These results provide a coherent working model for TIL origin, filiation and functional organization in primary NSCLC.