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Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non–small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. Implications: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.

Anti-PD-1/PD-L1 agents have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). To expand our understanding of the molecular features underlying response to checkpoint inhibitors in NSCLC, we describe here the first joint analysis of the Stand Up To Cancer-Mark Foundation cohort, a resource of whole exome and/or RNA sequencing from 393 patients with NSCLC treated with anti-PD-(L)1 therapy, along with matched clinical response annotation. We identify a number of associations between molecular features and outcome, including (1) favorable (for example, ATM altered) and unfavorable (for example, TERT amplified) genomic subgroups, (2) a prominent association between expression of inducible components of the immunoproteasome and response and (3) a dedifferentiated tumor-intrinsic subtype with enhanced response to checkpoint blockade. Taken together, results from this cohort demonstrate the complexity of biological determinants underlying immunotherapy outcomes and reinforce the discovery potential of integrative analysis within large, well-curated, cancer-specific cohorts.

Neoadjuvant ipilimumab + nivolumab (Ipi+Nivo) and nivolumab + chemotherapy (Nivo+CT) induce greater pathologic response rates than CT alone in patients with operable non-small cell lung cancer (NSCLC). The impact of adding ipilimumab to neoadjuvant Nivo+CT is unknown. Here we report the results and correlates of two arms of the phase 2 platform NEOSTAR trial testing neoadjuvant Nivo+CT and Ipi+Nivo+CT with major pathologic response (MPR) as the primary endpoint. MPR rates were 32.1% (7/22, 80% confidence interval (CI) 18.7–43.1%) in the Nivo+CT arm and 50% (11/22, 80% CI 34.6–61.1%) in the Ipi+Nivo+CT arm; the primary endpoint was met in both arms. In patients without known tumor EGFR/ALK alterations, MPR rates were 41.2% (7/17) and 62.5% (10/16) in the Nivo+CT and Ipi+Nivo+CT groups, respectively. No new safety signals were observed in either arm. Single-cell sequencing and multi-platform immune profiling (exploratory endpoints) underscored immune cell populations and phenotypes, including effector memory CD8+ T, B and myeloid cells and markers of tertiary lymphoid structures, that were preferentially increased in the Ipi+Nivo+CT cohort. Baseline fecal microbiota in patients with MPR were enriched with beneficial taxa, such as Akkermansia, and displayed reduced abundance of pro-inflammatory and pathogenic microbes. Neoadjuvant Ipi+Nivo+CT enhances pathologic responses and warrants further study in operable NSCLC. (ClinicalTrials.gov registration: NCT03158129.)

Purpose: Patients with advanced non–small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. Experimental Design: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. Results: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell–mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. Conclusions: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

The phase II DETERRED trial assessed the safety and efficacy of consolidation and concurrent immunotherapy with chemoradiation in unresectable locally advanced non-small cell lung cancer. We present updated efficacy analysis of this trial.The trial was conducted in 2 parts with patients in part 1 (n = 10) receiving chemoradiation with consolidation atezolizumab, while patients in part 2 (n = 30) received concurrent and consolidation atezolizumab. Progression-free survival (PFS), time to second progression (PFS2), and overall survival (OS) were assessed using Kaplan-Meier analysis. Subset analyses were performed by programmed cell death ligand-1 (PD-L1) status and targetable driver oncogene mutation status.At a median follow-up of 39.2 months, the median PFS for part 1 was 18.9 months and 15.1 months for part 2. Median OS for part 1 was 26.5 months and was not reached for part 2. For the cohort, 3-year OS was 53.8%, while 4-year OS was 47.4%. Patients with targetable driver oncogene mutations had a median PFS of 9.4 months and OS of not reached compared to 16.6 months (HR: 3.49, p = 0.02) and 26.9 months (HR: 0.40, p = 0.12) respectively compared to those without targetable driver oncogene mutations. Patients with PD-L1 1% had median PFS of 11.0 months and OS of 26.5 months compared to 27.4 months (HR: 2.01, p = 0.10) and not reached (HR: 1.49, p = 0.41) respectively for those with PD-L1 ≥ 1%.In the DETERRED trial, chemoradiation with concurrent and/or consolidative atezolizumab led to comparable efficacy as consolidative durvalumab in the PACIFIC trial. The presence of targetable driver oncogene mutations led to worse PFS, while PD-L1 1% trended to worse PFS.

PURPOSE Advances in biological measurement technologies are enabling large-scale studies of patient cohorts across multiple omics platforms. Holistic analysis of these data can generate actionable insights for translational research and necessitate new approaches for data integration and mining. METHODS We present a novel approach for integrating data across platforms on the basis of the shared nearest neighbors algorithm and use it to create a network of multiplatform data from the immunogenomic profiling of non–small-cell lung cancer project. RESULTS Benchmarking demonstrates that the shared nearest neighbors-based network approach outperforms a traditional gene-gene network in capturing established interactions while providing new ones on the basis of the interplay between measurements from different platforms. When used to examine patient characteristics of interest, our approach provided signatures associated with and new leads related to recurrence and TP53 oncogenotype. CONCLUSION The network developed offers an unprecedented, holistic view into immunogenomic profiling of non–small-cell lung cancer, which can be explored through the accompanying interactive browser that we built.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

Potential ZEB1 acetylation-dependent target genes.

qPCR Primer Sequences

Antibody information

S1. K811 acetylation is integral to ZEB1 molecular weight. S2. &tilde225 kDa ZEB1 band regulates protein stability. S3. K811 acetylation facilitates ZEB1/NuRD interaction. S4. K811 acetylation directs the ZEB1/NuRD complex to bind at genomic promoters. S5. K811 acetylation decreased epithelial expression levels. S6. RNA-seq reveals K811 acetylation downregulates the expression of epithelial genes. S7. K811 acetylation in ZEB1 KO cells promotes lung adenocarcinoma migration and invasion. S8. K811 acetylation enhances metastatic potential of murine NSCLC cells.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non–small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition.Implications:The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.

Protein expression values of the immunoprecipitated samples

ChIP Primer Sequences

Introduction Immune checkpoint inhibitor (ICI) pneumonitis causes substantial morbidity and mortality. Estimates of real-world incidence and reported risk factors vary substantially. Methods We conducted a retrospective review of 419 patients with advanced non-small cell lung cancer (NSCLC) who were treated with anti-PD-(L)1 with or without anti-CTLA-4 therapy. Clinical, imaging, and microbiological data were evaluated by multidisciplinary adjudication teams. The primary outcome of interest was grade ≥2 (CTCAEv5) pneumonitis. Clinicopathologic variables, tobacco use, cancer therapies, and preexisting lung disease were assessed for univariate effects using Cox proportional hazards models. We created multivariate Cox proportional hazards models to assess risk factors for pneumonitis and mortality. Pneumonitis, pneumonia, and progression were modeled as time-dependent variables in mortality models. Results We evaluated 419 patients between 2013 and 2021. The cumulative incidence of pneumonitis was 9.5% (40/419). In a multivariate model, pneumonitis increased the risk for mortality (HR 1.6, 95% CI, 1.0-2.5), after adjustment for disease progression (HR 1.6, 95% CI, 1.4-1.8) and baseline shortness of breath (HR 1.5, 95% CI, 1.2-2.0). Incomplete resolution was more common with more severe pneumonitis. Interstitial lung disease was associated with higher risk for pneumonitis (HR 5.4, 95% CI, 1.1-26.6), particularly in never smokers (HR 26.9, 95% CI, 2.8-259.0). Conclusion Pneumonitis occurred at a high rate and significantly increased mortality. Interstitial lung disease, particularly in never smokers, increased the risk for pneumonitis.

Molecular modifiers of KRAS G12C inhibitor (KRAS G12Ci) efficacy in advanced KRAS G12C-mutant NSCLC are poorly defined. In a large unbiased clinico-genomic analysis of 424 NSCLC patients, we identified and validated co-alterations in KEAP1, SMARCA4 and CDKN2A as major independent determinants of inferior clinical outcomes with KRAS G12Ci monotherapy. Collectively, co-mutations in these three tumor suppressor genes segregated patients into distinct prognostic subgroups and captured ~50% of those with early disease progression (PFS≤3 months) with KRAS G12Ci. Pathway-level integration of less prevalent co-alterations in functionally related genes nominated PI3K/AKT/MTOR pathway and additional baseline RAS gene alterations, including amplifications, as candidate drivers of inferior outcomes with KRAS G12Ci, and revealed a possible association between defective DNA damage response/repair and improved KRAS G12Ci efficacy. Our findings propose a framework for patient stratification and clinical outcome prediction in KRAS G12C-mutant NSCLC that can inform rational selection and appropriate tailoring of emerging combination therapies.

Supplementary Fig S8 and legend

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319–29. ©2017 AACR.

Figure Legends for Supplementary Figures S1-S4.

Table 1: Pathology classification criteria. Table 2: Flow cytometry antibodies

Supplementary Fig. S1: A subset of treatment-naïve NSCLC and LCNEC tumors expresses high AXL levels and EMT scores.

IL-6 modulates expression of NK receptor and their ligands in EGFR mutant tumors.

A Matrigel surface was seeded first with GFP-tagged aggregates followed 24 h later by a CAF single-cell suspension. Time-lapse microscopy was immediately after CAF seeding. Scale bar, 200 μm. Movie compiled at 8 frames per second.

Supplementary Figures S10-S20 and figure legends

Top 200 genes by KS.

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We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1–knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting.Prevention Relevance:PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.

This file contains supplementary materials and methods; figures and figure legends and Tables.

Genes differentially expressed between IgG control and anti-PD-L1 treatment.

Supplemental Figure S1. Number of predicted neoantigens. Supplemental Figure S2. Association between expressed branch predicted neoantigens and relapse. Supplemental Figure S3. Substantial ITH in the number of unique T cell rearrangements. Supplemental Figure S4. Substantial ITH in tumor-enriched T cell repertoire in 8 patients whose blood samples were available. Supplemental Figure S5. A minority of T cell clones are shared between different regions within a tumor. Supplemental Figure S6. The top 5 clones within each tumor are conserved across all regions of that given tumor. Supplemental Figure S7. Tumor-enriched MOI in patients with available paired blood. Supplemental Figure S8. CD4-dominant T cell infiltrate in localized lung adenocarcinomas by immunohistochemical (IHC) staining. Supplemental Figure S9. Correlation between predicted neoantigen burden and TCR metrics. Supplemental Figure S10. Association between intratumor TCR heterogeneity and patient relapse. Supplemental Figure S11. Follow up time (months) in patients who recurred and those who have not. n.s., not significant.

Supplementary Figure S1. Lung cancer cell lines do not express NE and P3 gene transcripts. Supplementary Figure S2. CTL proliferate in the presence of NE-exposed lung cancer cells. Supplementary Figure S3. HLA class I presentation of mutant neoantigens is enhanced by NE and P3. Supplementary Figure S4. Immunoproteasome expression by lung cancer cells is unaltered by serine proteases.

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Supplementary Methods, Figure Legends

Supplementary Figure S1. Generation of GEMM-derived cell lines. Supplementary Figure S2. QPCR and Western blot analyses of DTYMK expression in 634 cells upon indicated shDtymks transduction. Supplementary Figure S3. Lkb1-wt 634 and Lkb1-null t4 cells transduced with shGFP, shDtymk-1 or shDtymk-3 and then cultured with or without additional 100 microM dTTP in medium for 4 days. Supplementary Figure S4. Lkb1-wt (634, 855, and 857) and Lkb1-null (t2, t4, and t5) cell lines (A) and human LKB1-wt (H358 and Calu-1) and LKB1-deficient H2122 and A549) NSCLC cell lines (B) were treated with CHEK1 inhibitors, AZD7762 and CHIR124, for the indicated dose and time, and then lysed for Western blot analysis of gammaH2AX. Supplementary Figure S5. Immunohistochemical analyses assessing gammaH2AZ. Supplementary Figure S6. QPCR and Western blot analyses of DTYMK expression in LKB1-wt Calu-1 NSCLC cells. Supplementary Figure S7. A schematic model is proposed based on our current data. Supplementary Figure S8. QPCR analyses of Dtymk (A) and Chek1 (B) transcript levels in Lkb1-wt (634, 855, and 857) and Lkb1-null (t2, t4, and t5) cell lines. Supplementary Figure S9. QPCR analysis of TK1 expression in 634 cells upon the indicated shTK1 transduction for 3 days. Supplementary Figure S10. Gene expression levels of DTYMK in normal lung vs. lung adenocarcinoma from the Oncomine Cancer Microarray database.

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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

A Matrigel surface was seeded first with GFP-tagged aggregates followed 24 h later by a CAF single-cell suspension. Time-lapse microscopy was initiated 24 hours after CAF seeding. Dissociating tumor aggregates migrate toward each other (arrow). Scale bar, 100 μm. Movie compiled at 2 frames per second.

Percentage of CD4+CD44hi

Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used KrasLA1 mice, which develop lung adenocarcinomas from somatic activation of a KrasG12D allele. The lung tumors in KrasLA1 mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde–derived collagen cross-links (HLCC) and lower lysine-aldehyde–derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration.Implications: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells. Mol Cancer Res; 14(3); 287–95. ©2015 AACR.

Supplementary Methods