Non-small Cell Lung Cancer Tumor Progression Research Articles

Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer

BackgroundMajor histocompatibility complex class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and...

HTR3A Promotes Non-small Cell Lung Cancer Through the FOXH1/Wnt3A Signaling Pathway.

5-Hydroxytryptamine receptors (5-HTRs) are strongly correlated with tumor progression in various types of cancer. Despite this, the underlying mechanisms responsible for the role of 5-HTRs in non-small cell lung cancer (NSCLC) remains unclear. This study aimed to investigate the relationship between 5-hydroxytryptamine receptor 3A (HTR3A) and NSCLC development. Our findings indicated a higher distribution of HTR3A expression in NSCLC tissues when compared with normal tissues, where patients with high HTR3A levels demonstrated shorter overall survival times. In vitro analyses revealed that overexpression of HTR3A facilitated the proliferation and migration of NSCLC cell lines (A549 and NCI-H3255). Similarly, a notable acceleration of tumor growth and enhanced pulmonary tumorigenic potential were observed in HTR3A-overexpressing tumor-bearing mice. Mechanistically, upregulation of Forkhead Box H1 (FOXH1) by HTR3A led to the activation of Wnt3A/β-catenin signaling pathways, thereby promoting the development of NSCLC. Our report thus highlights the significance of the HTR3A/FOXH1 axis during tumor progression in NSCLC, proposing HTR3A as a possible diagnostic indicator and candidate target for clinical therapy.

Spatial colocalization and combined survival benefit of natural killer and CD8 T cells despite profound MHC class I loss in non-small cell lung cancer.

MHC class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. We used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFNγ-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis. Spatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (DFS) (HR=0.58, p=0.064) and overall survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1×10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFNγ+/- NK cells and T cells. We discovered that both IFNγ+ NK and CD8 T cells were more frequently associated with other IFNγ+ lymphocytes in comparison to IFNγ- NK cells and CD8 T cells (p<1×10-30). Moreover, IFNγ+ lymphocytes were most often found clustered near MHC-I+ tumor cells. Tumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Co-association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFNγ. Frequent co-localization of IFNγ+ NK cells with other IFNγ+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated.

Abstract 4759: Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer

Abstract Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients are associated with clinical benefit in the metastatic and adjuvant settings when treated with EGFR inhibitors, such as osimertinib. Despite its dramatic efficacy, most patients are partial responders and refractive disease leave limited treatment options. The objective was to dissect the mechanism of osimertinib resistance in EGFR mutant NSCLC models to identify a means of re-sensitization. Imaging mass cytometric analysis of 76 NSCLC patients demonstrated EGFR expression was inversely correlated to CD105 membrane expression. CD105 is a transforming growth factor beta (TGF-β) family co-receptor that promotes bone morphogenic protein (BMP) signaling and inhibits TGF-β signaling. Significant elevation in cell surface CD105 by osimertinib was furthered by EGFR knockdown in two NSCLC lines with EGFR activating mutations. These lines were subjected to a program of increasing osimertinib concentrations to generate isogenic resistant lines. Heterogeneous drug-tolerant persister cells had significantly higher mutational load, increased CD105 cell surface expression as well as greater expression of bypass signaling factors such as ERK, PI3K, and BMP ligands compared to their parental counterparts. Single cell RNA-seq analysis of parental NSCLC revealed a small population of cells that shared elevated endothelial and pyrimidine metabolism, with a slow-cycling signature that was enriched in the osimertinib-resistant cells. Combining osimertinib with a CD105 neutralizing antibody, carotuximab, reduced the slow-cycling population and restored osimertinib sensitivity in resistant cell lines. The knockdown of CD105 had a more pronounced decrease in cell viability when combined with osimertinib. The osimertinib-resistant lines demonstrated more condensed chromatin and glycolytic state compared to the respective parental lines by ATAC-seq and Single Cell ENergetIc metabolism profiling, respectively. The addition of carotuximab reversed the chromatin and metabolic reprograming of osimertinib resistance. Carotuximab was discovered to initiate CD105 interaction with EGFR and its dominant active variant, EGFRv3. Ultimately, combination therapy of carotuximab and osimertinib resulted in significantly reduced tumor expansion compared to mice treated with either drug alone. Paradoxically, the small tumors from combination therapy had greater mitosis compared to the larger tumors of the osimertinib single agent treated mice. Inhibition of CD105 with carotuximab can overcome resistance to osimertinib and inhibit NSCLC tumor progression. This is a first-in-class pre-clinical foundation for a novel synthetic lethality treatment strategy resulting from the observation of global changes in EGFR-antagonist resistance as opposed to individual mutations identified in tumor subpopulations. Citation Format: Manish Thiruvalluvan, Sandrine Billet, Zhenqiu Liu, Joseph Lownik, Gabrielle Gonzales, Hyoyoung Kim, Anton L. Villamejor, Larry Milshteyn, Kamya Sankar, Edwin M. Posadas, Jean Lopategui, Sungyong You, Karen Reckamp, Neil A. Bhowmick. Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4759.

Long noncoding RNA lnc-SNAPC5-3:4 inhibits malignancy by directly upregulating miR-224-3p in non-small cell lung cancer

The mounting body of evidence demonstrates the growing importance of long noncoding RNAs in the advancement of tumors. This study aimed to investigate the molecular mechanism of lnc-SNAPC5-3:4 in non-small cell lung cancer (NSCLC). We investigated the expression of miR-224-3p and lnc-SNAPC5-3:4 in clinical NSCLC samples and NSCLC cell lines using reverse transcription polymerase chain reaction (RT-PCR). In vitro studies, A549 cell growth was estimated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EDU), and flow cytometry assays. In vivo studies, NSCLC tumorigenesis was determined using xenograft tumor mouse models, tumor growth was evaluated using antigen Kiel 67 (Ki67) staining, and tumor apoptosis was detected through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The relationship between lnc-SNAPC5-3:4 and miR-224-3p was determined by luciferase reporter gene assay. Results indicated that the expression of lnc-SNAPC5-3:4 was observed to be downregulated in NSCLC tissues and cell lines. After overexpression of lnc-SNAPC5-3:4 in cultured A549 cells, proliferation decreased and apoptosis increased. Furthermore, the expression of miR-224-3p was targeted and negatively regulated by lnc-SNAPC5-3:4. The lnc-SNAPC5-3:4 upregulation inhibited cell proliferation and promoted apoptosis, which was partially blocked by miR-224-3p overexpression in A549 cells. In addition, we constructed a subcutaneous inoculation model using BALB/c nude mice, and the results indicated that lnc-SNAPC5-3:4 overexpression restrained the growth of subcutaneous tumors, decreased Ki67 expression levels, and increased apoptosis, as indicated by TUNEL staining in nude mice. However, miR-224-3p transfection resulted in the reversal of the inhibitory effect of lnc-SNAPC5-3:4 on tumor growth. In conclusion, our study revealed that lnc-SNAPC5-3:4 inhibits tumor progression in NSCLC by targeting miR-224-3p. This study provides a potential therapeutic target for inhibiting NSCLC progression.

Determining the methylation status of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in patients with non-small cell lung cancer

The aim of this study was to determine the methylation status of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue in patients with non-small cell lung cancer (NSCLC). A relative level of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes was determined by the quantitative methylation-specific PCR in 73 patients with NSCLC. The quantitative methylation-specific reaction was performed both for tumor tissue samples and non-tumor tissue samples of the same patient. For each of the samples, a reaction was set both by the investigated genes (MARCH11, UNCX, HOXA9, and PTGDR) and by the reference beta-actin gene (β-actin). Positive levels of methylation of the HOXA9 gene were established for 83.5 % patients; the MARCH11 gene – for 80.8 % patients; the PTGDR gene – for 68.4 % patients; the UNCX gene – for 84.9 % patients. In the study group of patients with NSCLC, significant differences were found in the relative levels of methylation of the promoter regions of MARCH11, HOXA9, PTGDR, and UNCX genes in the tumor and non-tumor lung tissue. The data suggest that hypermethylation of MARCH11, HOXA9, PTGDR, and UNCX genes may play a role in NSCLC tumor progression.

Loss of BLK expression as a potential predictor of poor prognosis and immune checkpoint blockade response in NSCLC and contribute to tumor progression

BackgroundImmune checkpoint blockade (ICB) has been proved to have significant anti-tumor effect in the clinical treatment of non-small cell lung cancer (NSCLC). Therefore, biomarkers predicting ICB response can provide better treatment for patients with NSCLC. MethodsDifferential expression genes (DEGs) were identified by ImmuCellAI database. Copy number alteration (CNA) was analyzed by cBioPortal. The predicted efficiency of 4 genes on cancer immunotherapy was assessed by ROC analysis. The survival value of BLK was analyzed by Kaplan-Meier plotter and Prognoscan analysis. Clinical significance of BLK IHC-TMA score in NSCLC was also explored. The CCK-8 assay, wound healing assay, western blot assay in vitro and subcutaneous xenograft experiments in vivo were used for investigating the functions of BLK. The RNA-sequencing were performed to screen BLK regulated genes and conducted for GO/KEGG enrichment analysis. The transcriptional regulatory factor of BLK promoter region was predicted by ChIP-seq analysis. Results39 common DEGs between ICB Response (R) group and No Response (NR) group with NSCLC were identified, in which the CNA frequency of BLK deletion (> 6%) was found. The predicted efficiency of BLK on immunotherapy was performed best in NSCLC (AUC>0.7). Low expression of BLK was related to NSCLC with significantly poor prognosis. BLK overexpression can inhibit growth of NSCLC via activating apoptosis pathway, inhibiting the G2M checkpoint and Glycolysis pathway. The enrichment analysis indicated that BLK regulated genes related to oncogenic potential in NSCLC. Besides, BLK expression was inhibited via H3K27me3 modification in A549 and H1299 cells. BLK mRNA level was negatively correlated with methylation and positively correlated with the tumor purity in NSCLC. ConclusionOur study provides strong evidence that low expression of BLK may serve as a biomarker for poor prognosis in NSCLC, while response to ICB therapy and contributes to NSCLC tumor progression.

Abstract 428: The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression

Abstract Background: Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. EGFR-TKI resistance may be mediated by epithelial-mesenchymal transition (EMT), which causes a motile and invasive phenotype similar to mesenchymal cells. Sphingosine kinase 2 (SphK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and increases cell proliferation, tumor survival and cell migration. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SphK2. However, the role of SphK2 in TKI resistance and EMT is not well understood. Hypothesis: We hypothesize that the inhibition/downregulation of SphK2 will reverse EMT, reduce tumorigenicity and overcome ER and OR resistance. Study design and Results: We studied expression of SphK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. In drug resistant cells, qPCR and immunoblotting studies showed that SphK2 was upregulated by 1.5-1.7 and 1.5-3.3 fold in ER and OR resistant cell lines respectively compared to parental cells. 48 hours after SphK2 knockdown with siRNA treatment, SphK2 was downregulated by 4.6-fold and EMT mediator E-cadherin expression was upregulated by 2.9-fold. EMT proteins Vimentin and N-cadherin were downregulated by 3.8 and 2.2 fold respectively (p≤0.01). Using an MTT assay, differences in percentage viability of cells with different treatments were calculated with respect to their viability in the presence of OR. We found that 81.43% of H358OR and 76.35% of the H3255OR cells were inhibited by a combination of Opaganib, a Sphk2 inhibitor, and OR, this is 27.74% and 12.28% higher compared to the additive effect of drug and inhibitor in H358OR and H3255 cells respectively. Furthermore, transwell migration assays, wound healing assays and spheroid assays were performed in OR resistant cells and the results indicated inhibition of the above assays by 94.97%, 82.36%, 88.67% respectively when treated with a combination of Opaganib and OR as compared to the diluent. This indicates the potential of SphK2 inhibition to overcome TKI resistance and EMT. To analyze the role of SphK2 expression in cancer progression, IHC was performed on 63 early and 48 late-stage lung cancer patient sections. We observed upregulation of SphK2 in late-stage lung cancer (p≤0.05), indicating its role in cancer progression. Conclusion: In this study, SphK2 was shown to decrease tumor survival, cancer cell proliferation, and expression of EMT markers. Thus, SphK2 may be a promising clinical target for overcoming EGFR-TKI resistance and NSCLC tumor progression. Citation Format: S M Nasifuzzaman, Namrata Dube, Katie Lam, Robert J. Hillwig, Neelu Puri. The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 428.

Abstract 6192: MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC

Abstract Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide, accounting for &amp;gt;2.2 million of new cancer diagnoses and 1.8 million cancer-related deaths in 2020. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, including adenocarcinomas and squamous cell carcinomas. Many lung adenocarcinomas have activating driver mutations in KRAS (~32% tumors), often co-occurring with mutations inactivating the TP53 tumor suppressor. Expression of the RNA-binding protein Musashi-2 (MSI2) MSI2 regulates target mRNA translation, and its expression is progressively elevated along with tumor stage in lung cancers. We previously showed that MSI2 actively promotes lung cancer growth, invasion, and metastasis, in part through promoting expression and activity of the TGFβR1/SMAD3 and EGFR signaling cascades. We now find that genetic MSI2 deficiency in a murine lung tumor model, Krasmut/Trp53KO/Msi2KO (KPM2) decreases both the total tumor number and tumor burden in comparison to control Krasmut/Trp53KO (KP) mice. Using a panel of KPM2 and KP cell lines we established from tumors from these mice, we found KPM2 cell lines demonstrate a significant decrease in proliferation compared to KP cell lines. Interestingly, reverse phase protein array (RPPA) comparison analysis of KPM2 and KP cell lines showed that MSI2 strongly and positively regulates expression of Ataxia-Telangiectasia Mutated (ATM) protein, with lower levels observed in KPM2 cells. The ATM serine/threonine protein kinase is a key transducer of DDR signaling in cases of double-strand breaks; ATM activation causes G1/S cell cycle arrest, contributes to efficiency of DNA repair, and triggers apoptosis in cases of repair failure. Notably, KPM2 cells showed significantly higher levels of γH2AX, suggesting inefficient DDR. Finally, untreated or cisplatin-treated KPM2 cells demonstrated G2/M arrest and had increased level of apoptosis as demonstrated by increase in caspase 3 cleavage, consistent with ATM loss and compensatory activation of Ataxia-telangiectasia and Rad3-related protein (ATR). Taken together, our data suggests that one of the consequences of MSI2 upregulation in NSCLC includes maintenance of an efficient DDR. Targeting MSI2 and its downstream signaling would be of therapeutic interest. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Rajendra P. Pangeni, Christopher Lengner, Evgeny Izumchenko, Jyoti Patel, John Karanicolas, Petr Makhov, Yanis Boumber. MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6192.

Abstract 6040: The CHST11-CP-iron axis contributes to NSCLC stemness

Abstract The emerging advances in cancer therapies have changed the landscape for the treatment, but there are limited treatments options for advanced non-small-cell lung cancer (NSCLC). Cancer stem cell-induced chemoresistance and tumor metastasis accounts for treatment failure and recurrence in NSCLC. Additionally, the up-regulated glycosaminoglycan-chondroitin sulfate (CS) biosynthesis pathway contributes to the mesenchymal state of lung cancer and related to unfavorable prognosis. Previously, we found carbohydrate sulfotransferase 11 (CHST11), a key CS biosynthetic enzyme, was associated with tumor progression and metastasis in NSCLC as well as lung fibrosis. Furthermore, ceruloplasmin (CP) acted as a downstream effector of CHST11 through interferon-γ signaling pathway stimulation and increased NSCLC metastatic features. However, the linkage between metabolic dysregulation and NSCLC stemness remains unclear. Here, we found not only overexpression of CHST11 but also CS-A treatment, the metabolite of CHST11, promoted NSCLC tumor sphere formation, the expression of cancer stemness markers, and transcriptional activities of stemness-related pathways. Iron was a necessary ion in upregulating cancer stemness properties such as tumor-initiation, metastasis and chemoresistance. We found both CHST11 and iron enhanced NSCLC tumorigenesis. Moreover, iron chelation disrupted CHST11-CP axis induced-stemness. The combination of iron chelator with chemotherapies showed the synergistic effect on antitumor as well as anti-cancer stemness and further promoted apoptosis. Our findings implicate that the CHST11-CP-iron axis facilitates tumor progression and cancer stemness in NSCLC. A regimen that combines an iron chelator and chemotherapies targeting cancer stem cells will benefit for not only preventing NSCLC tumorigenesis but cancer recurrence. Citation Format: Li-Jie Li, Wei-Min Chang, Michael Hsiao. The CHST11-CP-iron axis contributes to NSCLC stemness. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6040.

Circular RNA, circular RARS, promotes aerobic glycolysis of non-small-cell lung cancer by binding with LDHA.

Accumulating evidence has highlighted the critical roles of circular RNAs (circRNAs) in non-small-cell lung cancer (NSCLC). This study aims to unveil the roles of circRARS (circular RARS) (hsa_circ_0001551) in NSCLC. Quantitative real-time PCR was used to determine the expression of circRARS in NSCLC tissues and cells. Kaplan-Meier analysis was used to determine the prognostic value of circRARS expression. CCK8, transwell, and wound healing assays were used to assess the proliferation, invasion, and migration abilities of NSCLC cells. RNA pull-down, cell fraction, glucose consumption, lactate production, and lactate dehydrogenase activity assays were conducted to explore the potential mechanisms of circRARS in NSCLC. circRARS is upregulated in NSCLC tissues and positively correlated with smoking status, lymph node metastasis, and higher tumor stages. NSCLC patients with high expression of circRARS have poor overall survival. Functional assays demonstrated that circRARS accelerated the proliferation, invasion, and migration of NSCLC cells in vitro. The cell fraction suggested that circRARS mainly accumulated in cytoplasm and the RNA pull-down assay showed lactate dehydrogenase (LDHA) could bind with circRARS. Furthermore, circRARS positively regulates LDHA activity and LDHA expression at the transcription level. Moreover, downregulated circRARS decreases glucose consumption and lactate production and compromises aerobic glycolysis in NSCLC cells. Finally, rescue assays showed circRARS could promote NSCLC cell proliferation by regulating LDHA activity. This study shows that circRARS can promote glycolysis and tumor progression in NSCLC by regulating LDHA.

Development of FAP-Targeted Chimeric Antigen Receptor NK-92 Cells for Non-Small Cell Lung Cancer.

Over the past two decades, great progress has been made in advancing the early detection and multimodal treatment of non-small cell lung cancer (NSCLC). However, overall cure rates and survival rates of NSCLC are still not satisfactory, and research into new therapies is needed. This study attempted to construct human Fibroblast Activation Protein-Chimeric Antigen Receptor Natural killer (NK)-92 cells (hFAP-CAR-NK-92 cells) and explore their potential therapeutic effects in NSCLC. Immunohistochemistry analysis was carried out to examine fibroblast activation protein (FAP) and Gasdermin E (GSDME) expression in clinical specimens of lung adenocarcinoma and squamous cell carcinoma tissue. Then the engineered hFAP-CAR-NK-92 cells efficiency was determined in vitro with lactate dehydrogenase (LDH) cytotoxicity assay and the cell morphology of A549, H226, and cancer-related fibroblast (CAF) was observed by electron microscopy. After the co-culture of target cells and effect cells, flow cytometry was employed for examining the CD107a expression in the effect cells, and western blotting was conducted for the cleavage levels of Caspase 3 and GSDME proteins in the target cells. The safety and efficacy of hFAP-CAR-NK-92 cells adoptive transfer immunotherapy in a tumor-bearing mouse were evaluated. Clinical studies have shown FAP positivity in patients with NSCLC. Compared with A549 or H226 cells alone, FAP expression was notably raised in A549+CAF cells or H226+CAF cells in nude mice, respectively (p < 0.05). The killing efficiency of K562 cells was not significantly different between hFAP-CAR-NK-92 and NK-92 cells (p > 0.05). The hFAP-CAR-NK-92 cells presented a higher killing efficiency against the hFAP-target (A549-hFAP, H226-hFAP and CAF-hFAP) cells than the NK-92 cells (p < 0.05). The degranulation of CD107a and cleavage levels of GSDME and Caspase 3 protein in the hFAP-CAR-NK-92 group were higher than those in the NK-92 group (p < 0.05). The 300 nM Granzyme B also induced pyroptosis in hFAP- or GSDME-positive cells (p < 0.05). In vivo experiments revealed that hFAP-CAR-NK-92 cells inhibited tumor progression of hFAP-positive NSCLC (p < 0.05). In this study, we successfully constructed hFAP-CAR-NK-92 cells and confirmed that hFAP-CAR-NK-92 cells could target hFAP-positive NSCLC to inhibit the progression of NSCLC by activating the Caspase-3/GSDME pyroptosis pathway.

CircRNA has_circ_0017109 promotes lung tumor progression via activation of Wnt/β-catenin signaling due to modulating miR-671-5p/FZD4 axis

IntroductionAccumulating evidence highlights the critical roles of circular RNAs (circRNAs) in the malignant progression of cancers. In this study, we investigated the expression pattern of a newly identified circRNA (hsa_circ_0017109) in non–small cell lung cancer (NSCLC), and examined its downstream molecular targets.MethodsQuantitative real-time PCR (qRT-PCR) and Western blotting (WB) were conducted to quantify gene and protein expression. In vitro functional assays such as colony formation assay, cell counting kit-8 (CCK-8) and flow cytometry were used to study cell proliferation and apoptosis. RNA pull-down assay, luciferase reporter assay and RNA immunoprecipitation were performed to validate molecular interaction. Mouse xenograft model of NSCLC cells was used to assess the role of circ_0017109 in tumorigenesis.ResultsCirc_0017109 was upregulated in NSCLC tumor samples and cells. Silencing circ_0017109 impaired cell proliferation and promoted apoptosis in NSCLC cells, and circ_0017109 knockdown suppressed in vivo tumorigenesis of NSCLC cells in mouse xenograft model. MiR-671-5p was identified as a target of circ_0017109, and circ_0017109 negatively impacted on miR-671-5p expression. MiR-671-5p downregulated FZD4 and dampened the activity of Wnt/β-catenin signaling pathway. Circ_0017109 modulated FZD4 expression by suppressing miR-671-5p activity.ConclusionsElevated circ_0017109 expression promotes tumor progression of NSCLC by modulating miR-671-5p/FZD4/β-catenin axis.

Costimulatory molecule-related lncRNA model as a potential prognostic biomarker in non-small cell lung cancer.

Costimulatory molecules have been demonstrated to exert essential roles in multiple cancers. However, their role in lung cancer remains elusive. Here, we sought to identify costimulatory molecule-related lncRNAs in non-small cell lung cancer (NSCLC) and establish a prognostic signature to predict the prognosis of patients with NSCLC. A total of 535 lung adenocarcinoma (LUAD) and 502 lung squamous cell carcinoma (LUSC) patients from the cancer genome atlas (TCGA) database were recruited. A novel costimulatory molecule-based lncRNA prognostic model was constructed using the least absolute shrinkage and selection operator (LASSO) algorithm to predict the overall survival. The Homo_sapiens.GRCh38 data set was set as a reference file for probe annotation. A total of 593 costimulatory molecule-related lncRNAs were extracted. After analysis, six costimulatory molecule-related lncRNAs (AC084859.1, AC079949.2, HSPC324, LINC01150, LINC01150, and AC090617.5) were screened. A prognostic model based on the six lncRNAs was established using systematic bioinformatics analyses. The prognostic model had a prognostic value in NSCLC patients. Furthermore, a prognostic nomogram was established based on clinical parameters and a risk-score model. Patients with different risk scores had considerably different tumor-infiltrating immune cells, somatic mutational loading, clinical outcomes, signaling pathways, and immunotherapy efficacy. In addition, LINC01137 was associated with unfavorable disease outcomes and fueled tumor progression in NSCLC. Taken together, our study demonstrated that a costimulatory molecule-related lncRNA model could be a potential prognostic biomarker in NSCLC. Moreover, LINC01137 could facilitate the proliferation and invasion of lung cancer.

Wnt2b and Wnt5a expression is highly associated with M2 TAMs in non‑small cell lung cancer.

Tumor-associated macrophages (TAMs), particularly M2 macrophages, promote tumor progression, while Wnt genes encode a family of multi-functional glycoproteins that serve an important role in tumorigenesis. Immunohistochemical studies were performed to evaluate Wnt2b and Wnt5a expression in tumor and stromal cells in M2 and M1 TAMs and Ki-67 proliferation index in 160 consecutive patients with resected non-small cell lung cancer (NSCLC). Overall, 52 tumors (32.5%) were classified as tumoral Wnt2b-high (Wnt2b-positive tumor cells >30%) and 95 (59.4%) as stromal Wnt2b-high (Wnt2b-positive stromal cells >30%), while 75 (46.9%) were classified as tumoral Wnt5a-high (Wnt5a-positive tumor cells >30%) and 63 (39.4%) as stromal Wnt5a-high (Wnt5a-positive stromal cells >28%). The density of M2 TAMs was significantly higher in the tumoral (P=0.0024) and stromal Wnt2b-high groups (P=0.0054). The density of M2 TAMs was also significantly higher in the tumoral (P=0.0005) and stromal Wnt5a-high groups (P=0.0486). By contrast, no difference in stromal or islet M1 TAM density was observed in relation to tumoral or stromal Wnt2b or Wnt5a status. Furthermore, Ki-67 proliferation index was significantly higher in the tumoral (P=0.0121) and stromal Wnt2b-high (P=0.0019) and tumoral Wnt5a-high (P=0.0088) groups. Overall survival rate was significantly lower in the Wnt2b-high (P=0.0437), Wnt5a-high (P=0.0106) and M2 TAM-high (P=0.0060) groups. Wnt2b and Wnt5a expression in tumor and stromal cells may induce M2 TAMs to produce more aggressive behavior during tumor progression in NSCLC.

Bojungikki-Tang Enhances the Effect of PD-1 Blockade in a Syngeneic Murine Model of Lung Carcinoma

Although immunotherapy has become the standard of treatment for non-small cell lung cancer (NSCLC), only a limited number of patients benefit from it clinically because of the resistance to immunotherapeutic agents. Of late, the usefulness of herbal medicines in combination with immunotherapy has been investigated. Bojungikki-Tang (BJIKT) is a widely used traditional herbal medicine. It synergistically enhances the antitumor effects of chemotherapy and regulates the immune responses in cancer, but its antitumor effect with immunotherapy in NSCLC is unclear. In this study, we investigated the combined effects of BJIKT and an anti-PD-1 antibody in a KLN205-DBA/2 syngeneic lung cancer model. Immunohistochemistry and flow cytometry analyses were performed to analyze the changes in immune cells in the tumor microenvironment. BJIKT plus an anti-PD-1 antibody treatment significantly inhibited tumor growth, unlike the respective monotherapies. Compared to monotherapy, the combination treatment resulted in a higher population of CD8+ cytotoxic T cells and a lower number of Ki67+ cells in the tumor tissues. Furthermore, the combination treatment decreased the proportion of myeloid-derived suppressor cells but increased the proportion of M1-like macrophages compared to that observed with monotherapy. Cytokine analysis showed that the combination treatment increased the levels of T helper type 1-related cytokines. Network pharmacology analysis revealed that BJIKT might regulate multiple signaling pathways related to immune function and tumor progression in NSCLC. These findings indicate that the combination treatment with BJIKT and an anti-PD-1 antibody effectively suppresses tumor growth by regulating immune function and may be an alternative therapeutic option for the treatment of NSCLC.

Abstract 874: MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC

Abstract Lung cancer holds second place in incidence and first place in mortality among all cancers, both in men and women, with metastasis contributing to the vast majority of deaths. Recently, expression of vascular endothelial growth factor receptor-2 (VEGFR2) on both endothelial and tumor cells was demonstrated as one of the key mechanisms contributing to growth of non-small cell lung cancer (NSCLC), the most common subtype of lung cancer. VEGFR2 dependent signaling is critical both for tumor autocrine functions related to NSCLC migration, and in paracrine control of tumor angiogenesis through action on tumor-proximal endothelial cells, suggesting a potential mechanism for growth control. Several drugs targeting VEGFR2 signaling pathway demonstrated their clinical efficacy in NSCLC setting, including VEGFR2 and VEGF-A inhibitors, VEGF-A/PIGF trap. Recently our group demonstrated that RNA-binding protein Musashi-2 (MSI2) is driving progression and metastasis of NSCLC. MSI2 regulates mRNA translation of multiple targets, its expression is upregulated in the metastasis-competent murine and human lung cancer cell lines and is progressively elevated in lung cancer samples. The goal of this study is to define the role of MSI2 in sustaining VEGFR2 signaling during NSCLC progression. Using reverse RNA immunoprecipitation (RIP) qPCR analysis, we demonstrate that MSI2 directly binds VEGFR2 mRNA. Reverse protein phase array (RPPA) of several NSCLC cell line models with expression or knockdown of MSI2 and subsequent direct validation showed that MSI2 strongly and positively regulates expression of VEGFR2. Subsequent mechanistical analysis revealed that MSI2 knockdown leads to decreased VEGFR2 protein and mRNA levels expression in several NSCLC cell lines in vitro, resulting in increased apoptosis via inhibition of VEGFR2 signaling pathway. To better characterize MSI2 biology, we’ve crossed the B6/129S4-Msi2tm1.1Cjl/J (M2) mice with B6/129S/Sv-Krastm3Tyj/J;Trp53tm1Brn/J (KP) mice and generated novel B6/129S/Sv-KP;Msi2-/- (KPM2) mouse model. Induction of tumorigenesis in KP mice using Cre adenovirus leads to development of lung adenocarcinoma. Induction of lung-specific Cre resulted in tumorigenesis in both KP and KPM2 mice. Interestingly, we observed significant decrease in both total lung tumor number and total lung tumor burden in KPM2 compared to KP mice. Next, we established three novel KP and KPM2 derived cell lines from mouse lung tumors and found that KPM2 cell lines demonstrate reduced proliferation capacity and VEGFR2 mRNA level relative to KP cell lines. Additional studies to understand an impact of MSI2 deletion in KP cell lines are ongoing. Taken together, MSI2 is a promising target for NSCLC treatment, as this protein regulates VEGFR2 signaling and apoptosis in cell models and contributes to KP tumorigenesis in vivo. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Petr Makhov, Alexander Kudinov, Anna Nikonova, John Karanicolas, Erica Golemis, Christopher Lengner, Hossein Borghaei, Jyoti Patel, Yanis Boumber. MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 874.

Abstract B020: Distinct spatiotemporal tumor-immune ecologies enable disease prediction in NSCLC patients

Abstract Purpose of study: Non-small cell lung cancer (NSCLC) is the most common and fatal of cancers. In this work, we examine multiplexed images of NSCLC tumors to investigate both the spatial and spatiotemporal eco-evolutionary interactions between the tumor and its microenvironment, to better understand NSCLC tumor progression and therapy response. Methods: We developed a scalable, computational image analysis pipeline using cell-segmentation and quadrat-based approaches to analyze the spatial and temporal features of high-dimensional multiplexed NSCLC images. Multiplexed images enable the spatial readouts of numerous biomarkers per tissue sample and allow the interpretation of cellular states and the characterization of tumor-immune interactions across tissue ensembles. We also implement statistical approaches for ecological niche modelling combined with machine learning and deep learning models to predict disease progression and identify clinical imaging biomarkers. Data: Images were obtained from two 9-patient cohorts having advanced/metastatic NSCLC who were treated with the oral HDAC inhibitor vorinostat combined with the PD-1 inhibitor pembrolizumab. The first cohort had 4 progressors (PD) and 5 with stable disease (SD). The second cohort had 3 patients each in the PD, SD and partial response (PR) categories. Images were collected from all patients both pre- and on-treatment (during the third week). Results: Using our computational framework based on cell segments and quadrats, we confirm that different spatial neighborhoods exist that distinguish PD from SD, and that these spatial ecologies aid disease progression: PD patients have distinct ecologies with higher colocalization of PanCK+PD-1+FoxP3 indicating an immunosuppressive environment, whereas SD patients have a higher colocalization of PanCK+PD-L1 along with T cells, suggesting immunoactive tumor regions. These can be considered as potential biomarker candidates for predicting tumor progression. In an additional experiment where we include PR samples in our analyses, these distinct spatial neighborhoods are reinforced amongst PD, SD and PR patient groups corroborating the existence of spatiotemporal patterns. Further, we were able to predict treatment response with &amp;gt;91% accuracy given each patient’s spatial distribution of cell types from pre-treatment images. Using these predictions, we can generate risk maps at the patient level to identify areas of the tumor that are indicators of a higher probability of progression during treatment. Conclusions: We leveraged both single-cell and quadrat-resolution analysis of multiplexed imaging data and identified fundamentally distinct spatial ecologies between PD and SD patients. The ecology in PD patients appears to be primed for immune resistance even before treatment. This ecological diversity between SD and PD patients acts as a biomarker that enables accurate disease progression prediction. Our disease progression predictions can be used in conjunction with standard PD-L1 status to further strengthen personalized treatment strategies. Citation Format: Sandhya Prabhakaran, Chandler Gatenbee, Mark Robertson-Tessi, Amer A. Beg, Jhanelle Gray, Scott Antonia, Robert A. Gatenby, Alexander R.A. Anderson. Distinct spatiotemporal tumor-immune ecologies enable disease prediction in NSCLC patients [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B020.

Upregulated TIGIT+ and Helios+ regulatory T cell levels in bronchoalveolar lavage fluid of NSCLC patients

BackgroundThe tumour microenvironment reshapes the specific gene expression of regulatory T cells (Tregs). A better definition of Treg subpopulations in the non-small-cell lung cancer (NSCLC) milieu is expected to clarify the identity and functional mode of Tregs and lead to the identification of better therapeutic targets. MethodsA total of 53 peripheral blood (PB) samples from 36 NSCLC patients and 17 control subjects and 42 matched bronchoalveolar lavage fluid (BALF) samples from 31 NSCLC patients and 11 control subjects were obtained to examine the frequencies of Treg subgroups through flow cytometry. Fifteen PB samples from healthy individuals were collected to explore the differential functions of Treg subsets. The PB samples of 5 patients after chemotherapy were obtained to evaluate the effect of chemotherapy on Treg subsets. Serum CYFRA 21-1 levels in NSCLC patients were determined using an electrochemiluminescence immunoassay. ResultsThe proportions of CD4+CD25+FoxP3+ Tregs in both PB and BALF were increased in NSCLC patients compared to controls. In BALF, the TIGIT+, Helios+, and TIGIT+Helios+ Treg subset levels were significantly elevated; the levels of the last two subsets were associated with NSCLC development, while the level of TIGIT-Helios- Tregs was decreased. The proportions of overall Tregs and TIGIT+, Helios+, and Helios+TIGIT+ Tregs were positively correlated with the serum CYFRA 21-1 levels in all patients. Functional differences were observed between Helios+TIGIT+ and Helios-TIGIT- Tregs. After chemotherapy, regardless of the reduction in serum CYFRA 21-1 levels, the proportions of Tregs and Treg subsets did not change. ConclusionsElevated TIGIT+Helios+ and Helios+ Treg levels may play a role in NSCLC tumour progression, and targeting TIGIT and Helios on Tregs may be an effective treatment for NSCLC.

High miR-3648 expression and low APC2 expression are associated with shorter survival and tumor progression in NSCLC.

Emerging studies have demonstrated that microRNAs (miRNAs) play crucial roles in the carcinogenesis of many developing human tumors. However, the clinical significance and biological function of microRNA-3648 (miR-3648) in non-small cell lung cancer (NSCLC) have been largely undefined. The expression of miR-3648 and the mRNA of adenomatous polyposis coli 2 (APC2) in NSCLC tissues and cell lines were analyzed using quantitative real-time RT-PCR. The prognostic value of miR-3648 and APC2 was examined using the Kaplan-Meier method and Cox regression analyses. Experiments using NSCLC cells were conducted to explore the influences of miR-3648 on tumor cell proliferation, migration and invasion. Increased expression of miR-3648 was observed in NSCLC tissues and cell lines compared with the corresponding controls (all P<0.05). miR-3648 expression was associated with the differentiation, lymph node metastasis and TNM stage (all P<0.05) of NSCLC patients, and high expression of miR-3648 was associated with poor overall survival rate. NSCLC cell proliferation, migration and invasion were significantly enhanced by miR-3648 overexpression. The further luciferase reporter assay and expression results showed that the decreased APC2 might also be a prognostic biomarker, and served as a target of miR-3648 in NSCLC. The findings from the present study indicate that the overexpression of miR-3648 serves as a useful biomarker for the prediction of prognosis in NSCLC, and promotes tumor cell proliferation, migration and invasion. APC2, as another prognosis-related molecule, may be a target of miR-3648 in NSCLC.