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Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD.Significance:While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy.This article is highlighted in the In This Issue feature, p. 2483

Acknowledgements: We thank C. Zamilpa, D. Abeydeera and J. Collins, at UCLA’s Crump Imaging Technology Center, for assistance with PET–CT imaging of the mice. We thank the Translational Pathology Core Laboratory at UCLA’s DGSOM for assistance with tumour sample preparation and processing. We also thank M. McCaffery for assistance with electron microscopy imaging on in vitro-cultured cells and J. Castillo for assistance with graphics. This research was supported by the NIH National Center for Advancing Translational Science UCLA CTSI grant number UL1TR001881. D.B.S. was supported by the UCLA CTSI KL2 Translational Science Award grant number KL2TR001882 at the UCLA David Geffen School of Medicine, the UCLA Jonsson Comprehensive Cancer Center grant P30 CA016042, NIH/NCI R01 CA208642-01, American Cancer Society grant numbers RSG-16-234-01-TBG and MBG-19-172-01-MBG, and Department of Defense LCRP grant numbers W81XWH-13-1-0439 and W81XWH-18-1-0295. M.H. was supported by NIH/NCI R01 CA208642-01. A.L. was supported by grant NIH-NCI K08 CA245249-01A1 and a LUNGevity 2019 Career Development Award. M. Momcilovic was supported by American Cancer Society grant numbers RSG-16-234-01-TBG and MBG-19-172-01-MBG. A.G. was supported by an NIH/NCI R01 CA208642-01 diversity supplement. J.T.L. was supported by NIH/NCI P30 CA016042. D.M.W. is supported by EMBO long-term fellowship ALTF 828-2021. This research was supported by the Joyce and Saul Brandman Fund for Medical Research. We extend our heartfelt thanks and appreciation to the Scott family and the Carrie Strong Foundation as well as B. and D. Goldfarb for their generous support., Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

Supplementary Data from The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma

Supplementary Figure from The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma

Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD.Significance:While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Fig S8 and legend

Ample studies suggest that the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway plays a pivotal role in carcinogenesis and that COX-2 inhibition may help prevent lung cancer. Therefore, we conducted a randomized, double-blind, placebo-controlled trial of the COX-2–selective inhibitor celecoxib (400 mg bid for 6 months) in former-smokers (age ≥ 45, ≥ 30 pack-years of smoking, ≥ 1 year of sustained abstinence from smoking). We assessed the impact of celecoxib on cellular and molecular events associated with lung cancer pathogenesis; the primary endpoint was bronchial Ki-67 labeling index (Ki-67 LI) after 6 months of treatment. Of 137 randomized subjects, 101 completed both baseline and 6-month bronchoscopies and were evaluable for the primary endpoint analysis. The beneficial effect on Ki-67 LI was greater in the celecoxib arm (versus placebo) in a mixed-effects analysis (P = 0.0006), and celecoxib significantly decreased Ki-67 LI by an average of 34%, whereas placebo increased Ki-67 LI by an average of 3.8% (P = 0.04; t test). In participants who crossed over to the other study arm at 6 months (all of whom had received 6 months of celecoxib at the end of a 12 months treatment period), the decreases in Ki-67 LI correlated with a reduction and/or resolution of lung nodules on computed tomography. Celecoxib significantly reduced plasma c-reactive protein and interleukin-6 mRNA and protein and increased 15(S)-hydroxy-eicosatetraenoic acid levels in bronchoalveolar lavage (BAL) samples. The baseline ratio of COX-2 to 15-hydroxyprostaglandin dehydrogenase mRNA in BAL cells was a significant predictive marker of Ki-67 response to celecoxib (P = 0.002). Our collective findings support the continued investigation of celecoxib for lung cancer chemoprevention in former smokers at a low risk of cardiovascular disease. Cancer Prev Res; 4(7); 984–93. ©2011 AACR.

Supplementary Table S3

Supplemental Tables, Figures, and Methods

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of “metastatic inefficiency.” Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514–24. ©2017 AACR.See related editorial by Hynds and Janes, p. 491

XLSX file - 48KB, IPA biological functions analysis.

Supplementary Figures S10-S20 and figure legends

PDF file - 10K, miR-125a expression in human tissue (normal and premalignant atypical adenomatous hyperplasia lesions).

PDF file 113K, Table describing manufacturer information and conditions for antibodies used in western blotting. Milk refers to Blotto, non-fat dry milk (Santa Cruz Biotechnology), and BSA refers to Bovine Serum Albumin, Fraction V (Fisher Scientific)

This perspective on Varella-Garcia et al. (beginning on p. 447 in this issue of the journal) discusses the role of sputum-based biomarkers in the risk assessment and early detection of lung cancer. The importance of the detection of sputum epithelial chromosomal aneusomy by fluorescence in situ hybridization (FISH) as a potential risk or early-detection biomarker is discussed in the context of other biomarkers and models in lung carcinogenesis. The presently reported findings on FISH in sputum cells are an important contribution worthy of further investigation in defined clinical settings. Cancer Prev Res; 3(4); 420–3. ©2010 AACR.

PDF file - 37K, Stable knockdown of c-myc expression by shRNA in NSCLC cells.

PDF - 69K, Supplemental Figure Legend

XLSX file - 343KB, Genes associated with SCC carcinogenesis.

XLS file - 32KB, MYC activity prediction and its target genes.

Heat shock protein 90 (HSP90) is involved in protein folding and functions as a chaperone for numerous client proteins, many of which are important in non–small cell lung cancer (NSCLC) pathogenesis. We sought to define preclinical effects of the HSP90 inhibitor NVP-AUY922 and identify predictors of response. We assessed in vitro effects of NVP-AUY922 on proliferation and protein expression in NSCLC cell lines. We evaluated gene expression changes induced by NVP-AUY922 exposure. Xenograft models were evaluated for tumor control and biological effects. NVP-AUY922 potently inhibited in vitro growth in all 41 NSCLC cell lines evaluated with IC50 < 100 nmol/L. IC100 (complete inhibition of proliferation) < 40 nmol/L was seen in 36 of 41 lines. Consistent gene expression changes after NVP-AUY922 exposure involved a wide range of cellular functions, including consistently decreased dihydrofolate reductase after exposure. NVP-AUY922 slowed growth of A549 (KRAS-mutant) xenografts and achieved tumor stability and decreased EGF receptor (EGFR) protein expression in H1975 xenografts, a model harboring a sensitizing and a resistance mutation for EGFR-tyrosine kinase inhibitors in the EGFR gene. These data will help inform the evaluation of correlative data from a recently completed phase II NSCLC trial and a planned phase IB trial of NVP-AUY922 in combination with pemetrexed in NSCLCs. Mol Cancer Ther; 12(6); 890–900. ©2013 AACR.

PDF - 769K, Heat maps from microarray analyses of gene expression changes seen in at least 13 of 16 experiments after exposure to NVP-AUY922

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment.Significance:The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355

PDF file - 85KB

Supplementary Methods

PDF file - 690KB, Supplementary Figure S1. Laser capture microdissection. Supplementary Figure S2. Experimental validation of PTBP3 expression. Supplementary Table S1. Patient demographics. List of SCC patients with their age, gender, grade of premalignant lesion, and assays performed. Supplementary Table S2. Alignment statistics. The number and percentage of reads aligning uniquely to the nuclear (autosomal and sex) or mitochondrial chromosomes, or not uniquely aligned, are shown for each sample.

Supplementary Figures, Figure Legends, and Table Legends

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAFnon-V600, and BRAFV600 mutations. Tumor cell–intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti–PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy.Significance:Type I RAFi + MEKi are indicated only in certain BRAFV600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521

Definition of the molecular pathogenesis of lung cancer allows investigators an enhanced understanding of the natural history of the disease, thus fostering development of new prevention strategies. In addition to regulating epithelial-to-mesenchymal transition (EMT), the transcription factor Snail exerts global effects on gene expression. Our recent studies reveal that Snail is upregulated in non–small cell lung cancer (NSCLC), is associated with poor prognosis, and promotes tumor progression in vivo. Herein, we demonstrate that overexpression of Snail leads to the upregulation of secreted protein, acidic and rich in cysteine (SPARC) in models of premalignancy and established disease, as well as in lung carcinoma tissues in situ. Snail overexpression leads to increased SPARC-dependent invasion in vitro, indicating that SPARC may play a role in lung cancer progression. Bioinformatic analysis implicates transforming growth factor beta (TGF-β), extracellular signal–regulated kinase (ERK)1/2, and miR-29b as potential intermediaries in Snail-mediated upregulation of SPARC. Both the TGF-β1 ligand and TGF-β receptor 2 (TGF-βR2) are upregulated following Snail overexpression. Treatment of human bronchial epithelial cell (HBEC) lines with TGF-β1 and inhibition of TGF-β1 mRNA expression modulates SPARC expression. Inhibition of MAP–ERK kinase (MEK) phosphorylation downregulates SPARC. MiR-29b is downregulated in Snail-overexpressing cell lines, whereas overexpression of miR-29b inhibits SPARC expression. In addition, miR-29b is upregulated following ERK inhibition, suggesting a Snail-dependent pathway by which Snail activation of TGF-β and ERK signaling results in downregulation of miR-29b and subsequent upregulation of SPARC. Our discovery of pathways responsible for Snail-induced SPARC expression contributes to the definition of NSCLC pathogenesis. Cancer Prev Res; 7(1); 150–60. ©2013 AACR.

Understanding the molecular pathogenesis of lung cancer is necessary to identify biomarkers/targets specific to individual airway molecular profiles and to identify options for targeted chemoprevention. Herein, we identify mechanisms by which loss of microRNA (miRNA)125a-3p (miR125a) contributes to the malignant potential of human bronchial epithelial cells (HBEC) harboring an activating point mutation of the K-ras proto-oncogene (HBEC K-ras). Among other miRNAs, we identified significant miR125a loss in HBEC K-ras lines and determined that miR125a is regulated by the PEA3 transcription factor. PEA3 is upregulated in HBEC K-ras cells, and genetic knockdown of PEA3 restores miR125a expression. From a panel of inflammatory/angiogenic factors, we identified increased CXCL1 and vascular endothelial growth factor (VEGF) production by HBEC K-ras cells and determined that miR125a overexpression significantly reduces K-ras–mediated production of these tumorigenic factors. miR125a overexpression also abrogates increased proliferation of HBEC K-ras cells and suppresses anchorage-independent growth (AIG) of HBEC K-ras/P53 cells, the latter of which is CXCL1-dependent. Finally, pioglitazone increases levels of miR125a in HBEC K-ras cells via PEA3 downregulation. In addition, pioglitazone and miR125a overexpression elicit similar phenotypic responses, including suppression of both proliferation and VEGF production. Our findings implicate miR125a loss in lung carcinogenesis and lay the groundwork for future studies to determine whether miR125a is a possible biomarker for lung carcinogenesis and/or a chemoprevention target. Moreover, our studies illustrate that pharmacologic augmentation of miR125a in K-ras–mutated pulmonary epithelium effectively abrogates several deleterious downstream events associated with the mutation. Cancer Prev Res; 7(8); 845–55. ©2014 AACR.

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XLS file - 35KB, TP53 activity prediction and its target genes.

Aberrant expression of microRNAs (miRNA) with oncogenic capacities (oncomiRs) has been described for several different malignancies. The first identified oncomiR, miR-17-92, is frequently overexpressed in a variety of cancers and its targets include the tumor suppressor PTEN. The transcription factor c-Myc (MYC) plays a central role in proliferative control and is rapidly upregulated upon mitogenic stimulation. Expression of c-Myc is frequently deregulated in tumors, facilitating proliferation and inhibiting terminal differentiation. The c-Myc–regulated network comprises a large number of transcripts, including those encoding miRNAs. Here, prostaglandin E2 (PGE2) exposure rapidly upregulates the expression of the MYC gene followed by the elevation of miR-17-92 levels, which in turn suppresses PTEN expression, thus enhancing apoptosis resistance in non–small cell lung cancer (NSCLC) cells. Knockdown of MYC expression or the miR-17-92 cluster effectively reverses this outcome. Similarly, miR-17-92 levels are significantly elevated in NSCLC cells ectopically expressing COX-2. Importantly, circulating miR-17-92 was elevated in the blood of patients with lung cancer as compared with subjects at risk for developing lung cancer. Furthermore, in patients treated with celecoxib, miR-17-92 levels were significantly reduced. These data demonstrate that PGE2, abundantly produced by NSCLC and inflammatory cells in the tumor microenvironment, is able to stimulate cell proliferation and promote resistance to pharmacologically induced apoptosis in a c-Myc and miR-17-92–dependent manner.Implications: This study describes a novel mechanism, involving c-Myc and miR-17-92, which integrates cell proliferation and apoptosis resistance. Mol Cancer Res; 12(5); 765–74. ©2014 AACR.

Related Perspective and Related Article from Lung Cancer Biomarkers: FISHing in the Sputum for Risk Assessment and Early Detection

PDF file - 47K, Patient characteristics.

Perspective on This Article from Lung Cancer Chemoprevention with Celecoxib in Former Smokers