Abstract Background: Therapies targeting immune checkpoint have significantly transformed the treatment of several cancers, including lung cancer. However, only 20% of lung cancer patients show a response to immunotherapies. KRAS and EGFR mutations are significant factors in the development and progression of lung cancer and its response to immunotherapy. Hence, it's vital to understand intra-tumoral heterogeneity, the makeup of the Tumor Microenvironment (TME), and the cellular interactions in tumors with KRAS and EGFR mutations to uncover new insights. Methods: Lung cancer tissues from 200 patients were collected, focusing on KRAS and EGFR mutation statuses, specifically KRAS+/EGFR-, KRAS-/EGFR+, and KRAS-/EGFR-. For each patient, two samples were taken, resulting in a total of 400 tissue microarray (TMA) cores. These samples were imaged at single-cell resolution using the PhenoCycler®-Fusion system, which employed a 42-plex antibody panel targeting immune, tumor, proliferation, and apoptosis markers. The first step in the analysis was cell segmentation, performed using a fine-tuned deep learning model. This process successfully segmented a total of 2.16 million cells across the entire collection of samples. Following this, the protein expressions for each cell were calculated, and unsupervised clustering was performed, resulting in 36 distinct clusters, that were manually annotated into 14 cell phenotypes. The proportions of these cell phenotypes were then quantified for each of the 400 TMA cores and compared across the three mutation groups. The analysis also included the spatial proximity and cellular neighborhoods in relation to these mutation groups. Results: Quantitative analysis focusing on the relative abundance of cell types revealed that the ratio of tumor cells to immune cells was significantly lower in KRAS-/EGFR- tumors compared to those with KRAS+/EGFR- and KRAS-/EGFR+ mutations. Furthermore, the percentages of Regulatory T cells (Tregs) and M1 Macrophages were significantly higher in the KRAS+/EGFR- group compared to the KRAS-/EGFR+ group. Spatial proximity analysis indicated that in the KRAS-/EGFR+ group, M2 Macrophages were significantly closer to Cytotoxic T cells and Helper T cells compared to the distances observed in the other two groups. Moreover, neighborhood analysis identified 20 distinct cellular neighborhoods, each defined by specific cell-cell interactions. COX hazard analysis based on these cellular neighborhoods demonstrated notable variations among the three mutation groups, which are linked to differences in patient outcomes. Conclusions: Through single-cell spatial phenotyping, this work has shown that the spatial immune landscape of lung tumors is influenced by KRAS and EGFR mutation statuses. This presents a novel opportunity to enhance our understanding of the spatial structure of lung cancer and to identify more effective therapeutic targets. Citation Format: Rajender Nandigama, Bassem Ben Cheikh, Ning Ma, Jochen Wilhelm, Laura Klotz, Florian Eichhorn, Mark Kriegsmann, Marek Bartkuhn, Jamal Nabhanizadeh, Sascha Seidel, Thorsten Stiewe, Albrecht Stenzinger, Andreas Weigert, Mario Looso, Friedrich Grimminger, Werner Seeger, Soni Savai Pullamsetti, Niyati Jhaveri, Hauke Winter, Rajkumar Savai. Single-cell spatial landscape of the mutation-specific human lung tumor immune microenvironment [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 5508.

Blood vessels in tumors are often dysfunctional. This impairs the delivery of therapeutic agents to and distribution among the cancer cells. Subsequently, treatment efficacy is reduced, and dose escalation can increase adverse effects on non-malignant tissues. The dysfunctional vessel phenotypes are attributed to aberrant pro-angiogenic signaling, and anti-angiogenic agents can ameliorate traits of vessel dysfunctionality. However, they simultaneously reduce vessel density and thereby impede drug delivery and distribution. Exploring possibilities to improve vessel functionality without compromising vessel density in the tumor microenvironment, we evaluated transcription factors (TFs) involved in epithelial-mesenchymal transition (EMT) as potential targets. Based on similarities between EMT and angiogenic activation of endothelial cells, we hypothesized that these TFs, Snai1 in particular, might serve as key regulators of vessel dysfunctionality. In vitro, experiments demonstrated that Snai1 (similarly Slug and Twist1) regulates endothelial permeability, permissiveness for tumor cell transmigration, and tip/stalk cell formation. Endothelial-specific, heterozygous knock-down of Snai1 in mice improved vascular quality in implanted tumors. This resulted in better oxygenation and reduced metastasis. Notably, the tumors in Snai1KD mice responded significantly better to chemotherapeutics as drugs were transported into the tumors at strongly increased rates and more homogeneously distributed. Thus, we demonstrate that restoring vessel homeostasis without affecting vessel density is feasible in malignant tumors. Combining such vessel re-engineering with anti-cancer drugs allows for strategic treatment approaches that reduce treatment toxicity on non-malignant tissues.

Targeting the supportive tumor microenvironment (TME) is an approach of high interest in cancer drug development. However, assessing TME-targeted drug candidates presents a unique set of challenges. We develop a comprehensive screening platform that allows monitoring, quantifying, and ranking drug-induced effects in self-organizing, vascularized tumor spheroids (VTSs). The confrontation of four human-derived cell populations makes it possible to recreate and study complex changes in TME composition and cell-cell interaction. The platform is modular and adaptable for tumor entity or genetic manipulation. Treatment effects are recorded by light sheet fluorescence microscopy and translated by an advanced image analysis routine in processable multi-parametric datasets. The system proved to be robust, with strong interassay reliability. We demonstrate the platform's utility for evaluating TME-targeted antifibrotic and antiangiogenic drugs side-by-side. The platform's output enabled the differential evaluation of even closely related drug candidates according to projected therapeutic needs. Assessing tumour microenvironment-targeted drug candidates remains challenging. Here, the authors develop a comprehensive screening platform that allows for monitoring, quantifying, and ranking drug-induced effects in self-organizing, vascularized tumour spheroids.

In tumor therapy anti-angiogenic approaches have the potential to increase the efficacy of a wide variety of subsequently or co-administered agents, possibly by improving or normalizing the defective tumor vasculature. Successful implementation of the concept of vascular normalization under anti-angiogenic therapy, however, mandates a detailed understanding of key characteristics and a respective scoring metric that defines an improved vasculature and thus a successful attempt. Here, we show that beyond commonly used parameters such as vessel patency and maturation, anti-angiogenic approaches largely benefit if the complex vascular network with its vessel interconnections is both qualitatively and quantitatively assessed. To gain such deeper insight the organization of vascular networks, we introduce a multi-parametric evaluation of high-resolution angiographic images based on light-sheet fluorescence microscopy images of tumors. We first could pinpoint key correlations between vessel length, straightness and diameter to describe the regular, functional and organized structure observed under physiological conditions. We found that vascular networks from experimental tumors diverted from those in healthy organs, demonstrating the dysfunctionality of the tumor vasculature not only on the level of the individual vessel but also in terms of inadequate organization into larger structures. These parameters proofed effective in scoring the degree of disorganization in different tumor entities, and more importantly in grading a potential reversal under treatment with therapeutic agents. The presented vascular network analysis will support vascular normalization assessment and future optimization of anti-angiogenic therapy.

The analysis of peritumoral immune cells may help to identify patients that profit from further immunotherapy treatment after lung cancer surgery. We analyzed tumors from 174 patients and identified high levels of tumor infiltrating lymphocytes and M1-like tumor-associated macrophages as positive predictive factor whereas the abundance of regulatory T-cells was associated with poorer outcome in distinct histologic non-small-cell lung cancer subtypes.Introduction: The tumoral immune milieu plays a crucial role for the development of non-small-cell lung cancer (NSCLC) and may influence individual prognosis. We analyzed the predictive role of immune cell infiltrates after curative lung cancer surgery.Materials and methods: The tumoral immune-cell infiltrate from 174 patients with pN1 NSCLC and adjuvant chemotherapy was characterized using immunofluorescence staining. The density and distribution of specific immune cells in tumor center (TU), invasive front (IF) and normal tissue (NORM) were correlated with clinical parameters and survival data.Results: Tumor specific survival (TSS) of all patients was 69.9% at 5 years. The density of tumor infiltrating lymphocytes (TIL) was higher in TU and IF than in NORM. High TIL density in TU (low vs. high: 62.0% vs. 86.7%; p = .011) and the presence of cytotoxic T-Lymphocytes (CTLs) in TU and IF were associated with improved TSS (positive vs. negative: 90.6% vs. 64.7% p = .024). High TIL-density correlated with programmed death-ligand 1 expression levels >= 50% (p < .001). Multivariate analysis identified accumulation of TIL (p = .016) and low Treg density (p = .003) in TU as negative prognostic predictors in squamous cell carcinoma (p = .025), whereas M1-like tumor- associated macrophages (p = .019) and high programmed death-ligand 1 status (p = .038) were associated with better survival in adenocarcinoma.Conclusion: The assessment of specific intratumoral immune cells may serve as a prognostic predictor in pN1 NSCLC. However differences were observed related to adenocarcinoma or squamous cell carcinoma histology. Prospective assessment of the immune-cell infiltrate and further clarification of its prognostic relevance could assist patient selection for upcoming perioperative immunotherapies.

Background: COPD is a heterogeneous disease involving different lung compartments. Changes in extracellular matrix (ECM) components, including basement membrane (BM) proteins, are associated with the disease progression. The alteration in collagen IV (colIV), a major component of the BM, and the precise distribution of six distinct colIV α-chains remains relatively understudied. Therefore, spatial expression and distribution of ECM proteins such as, colIV could help with phenotypic stratification of COPD patients Aim: To identify differential expression and distribution of diverse ColIV chains in different lung compartments of COPD patients Methods: We performed multiplex immunofluorescence (mIF) for compartmental profiling of six type IV collagen α-chains combined with cell-type specific markers and analyzed publicly available single cell RNA sequencing (scRNA-seq) profiles (GSE136831) from donor and COPD lungs Results: mIF showed differential geographic distribution of six colIV α-chains in various compartments of donor lungs. This abundance was different as compared with COPD lungs. Similarly, RNAseq analysis revealed different cell-type specific enrichment patterns of α-chains. The alveolar region was enriched in colIV α2,a3 and a4 chains whereas the bronchiolar region of donor and COPD lungs was enriched in α2,3,4,5 and 6 chains. In the vascular EC region, the expression of colIV α1 and 2 chains was prominent. A significant difference in the expression of α3,a4,a5 and a6 chains was observed between donor and COPD lungs Conclusion: We identified a divergent ColIV α-chain expression signature between donor and COPD lung, which could be used in the future to define different COPD phenotypes

The present treatments for lung cancer include surgical resection, radiation, chemotherapy, targeted therapy, and immunotherapy. Despite advances in therapies, the prognosis of lung cancer has not been substantially improved in recent years. Chimeric antigen receptor (CAR)-T cell immunotherapy has attracted growing interest in the treatment of various malignancies. Despite CAR-T cell therapy emerging as a novel potential therapeutic option with promising results in refractory and relapsed leukemia, many challenges limit its therapeutic efficacy in solid tumors including lung cancer. In this landscape, studies have identified several obstacles to the effective use of CAR-T cell therapy including antigen heterogeneity, the immunosuppressive tumor microenvironment, and tumor penetration by CAR-T cells. Here, we review CAR-T cell design; present the results of CAR-T cell therapies in preclinical and clinical studies in lung cancer; describe existing challenges and toxicities; and discuss strategies to improve therapeutic efficacy of CAR-T cells.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first -line defense mechanisms against bacterial infections of the lung.

Taxanes are known to have a profound effect on endothelial cells and the vasculature even at low doses. Here, we show that taxanes, rather than being anti-angiogenic, function more as vascular disrupting agents (VDAs), although they exert a different mechanism of vascular permeabilization when compared to traditional VDAs such as combretastatins. In the tumor context, this VDA-effect leads to a rapid vascular collapse and acute hypoxia. Concomitant treatment with anti-VEGF drugs aggravates hypoxia by blocking vasculogenic rescue mechanisms. While this results in a strong growth-suppressing effect on the tumor, it also increases its invasiveness and metastatic potential. We demonstrate that combination of anti-angiogenic drugs with taxanes blocks tumor reperfusion, intensifies intravasation of circulating tumor cells (CTCs) and strongly increases metastasis. Anti-VEGF drugs are commonly applied in combination with cytotoxic drugs including taxanes. Our findings have significant implications for the clinical use of this drug combination.### Competing Interest StatementThe authors have declared no competing interest.

Ever since RNA sequencing of whole genomes and transcriptomes became available, numerous RNA transcripts without having the classic function of encoding proteins have been discovered. Long non-coding RNAs (lncRNAs) with a length greater than 200 nucleotides were considered as “junk” in the beginning, but it has increasingly become clear that lncRNAs have crucial roles in regulating a variety of cellular mechanisms and are often deregulated in several diseases, such as cancer. Lung cancer is the leading cause of cancer-related deaths and has a survival rate of less than 10%. Immune cells infiltrating the tumor microenvironment (TME) have been shown to have a great effect on tumor development with macrophages being the major cell type within the TME. Macrophages can inherit an inflammatory M1 or an anti-inflammatory M2 phenotype. Tumor-associated macrophages, which are predominantly polarized to M2, favor tumor growth, angiogenesis, and metastasis. In this review, we aimed to describe the complex roles and functions of lncRNAs in macrophages and their influence on lung cancer development and progression through the TME.

For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter "taste" substances are most probably initiated by tracheal brush cells (BC). Our single-cell RNA-seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca2+]i in BC and subsequent ACh-release. ACh-release is regulated in an autocrine manner. While the muscarinic ACh-receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca2+]i in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5- and M3R-mediated. We show that ACh-release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.

Solid tumors are complex organ-like structures that consist not only of tumor cells but also of vasculature, extracellular matrix (ECM), stromal, and immune cells. Often, this tumor microenvironment (TME) comprises the larger part of the overall tumor mass. Like the other components of the TME, the ECM in solid tumors differs significantly from that in normal organs. Intratumoral signaling, transport mechanisms, metabolisms, oxygenation, and immunogenicity are strongly affected if not controlled by the ECM. Exerting this regulatory control, the ECM does not only influence malignancy and growth of the tumor but also its response toward therapy. Understanding the particularities of the ECM in solid tumor is necessary to develop approaches to interfere with its negative effect. In this review, we will also highlight the current understanding of the physical, cellular, and molecular mechanisms by which the pathological tumor ECM affects the efficiency of radio-, chemo-, and immunotherapy. Finally, we will discuss the various strategies to target and modify the tumor ECM and how they could be utilized to improve response to therapy.

Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively rendered tumor vessels highly permeable and caused massive intratumoral hemorrhage. While these results established platelets as potential targets for antitumor therapy, their depletion is not a treatment option due to their essential role in hemostasis. Thus, a detailed understanding of how platelets safeguard vascular integrity in tumors is urgently demanded. Here, we show for the first time that functional inhibition of glycoprotein VI (GPVI) on the platelet surface with an antibody (JAQ1) F(ab)2 fragment rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion while not inducing systemic bleeding complications. The intratumor bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells, confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, JAQ1 F(ab)2-mediated GPVI inhibition increased intratumoral accumulation of coadministered chemotherapeutic agents, such as Doxil and paclitaxel, thereby resulting in a profound antitumor effect. In summary, our findings identify platelet GPVI as a key regulator of vascular integrity specifically in growing tumors and could serve as a basis for the development of antitumor strategies based on the interference with platelet function.

The potential of altering the tumor ECM to improve drug response remains fairly unexplored. To identify targets for modification of the ECM aiming to improve drug response and overcome resistance, we analyzed expression data sets from pre-treatment patient cohorts. Cross-evaluation identified a subset of chemoresistant tumors characterized by increased expression of collagens and collagen-stabilizing enzymes. We demonstrate that strong collagen expression and stabilization sets off a vicious circle of self-propagating hypoxia, malignant signaling, and aberrant angiogenesis that can be broken by an appropriate auxiliary intervention: Interfering with collagen stabilization by inhibition of lysyl oxidases significantly enhanced response to chemotherapy in various tumor models, even in metastatic disease. Inhibition of collagen stabilization by itself can reduce or enhance tumor growth depending on the tumor type. The mechanistical basis for this behavior is the dependence of the individual tumor on nutritional supply on one hand and on high tissue stiffness for FAK signaling on the other.

As in the systemic treatment of any disease, it is crucial for anti-cancer drugs to reach their target at a sufficient that is a therapeutically effective dose. However, unlike normal organs, solid tumors have a tendency to be undersupplied and hypoxic. This not only leads to insufficient supply of oxygen and nutrients but also to inefficient transport of drugs into tumors. As a consequence, administered doses have to be raised, resulting in increased side effects and often premature termination of treatment. A better understanding of the mechanisms that hamper transport of drugs into tumors could lead to the development of auxiliary strategies aimed at increasing tumor drug delivery and accumulation and thereby improving the efficacy of anti-cancer drugs at our disposal. The tumor microenvironment (TME), i.e., its vasculature, stroma, extracellular matrix and immune environment affect the transport of drugs to the tumor and their distribution within the tumor tissue in various ways. In this review we will highlight the current research regarding the cellular and molecular mechanisms that remain as an obstacle towards an effective cancer therapy, and also focus on the various strategies to alter the TME to increase tumor drug exposure and thereby treatment efficacy.

Neurotrophic factors can alter the functional properties of sensory C-fibers in allergic asthma. Neurturin is a neurotrophic factor in the Glial Cell Line-Derived Neurotrophic Factor (GDNF) family of ligands, binds to its receptor GFRα2 and signals via co-receptor RET. Previously, we were able to demonstrate that some bronchopulmonary vagal C-fibers co-express not only GFRα2 and RET but also transient receptor potential channel vanilloid 1 and ankyrin 1 (TRPV1 and TRPA1). These TRP-channels are activated by many environmental irritants that induce allergic reactions like dyspnea, cough, hypersecretion and bronchoconstriction in patients suffering from allergic asthma. Therefore it has been speculated that the dysfunction of the TRPV1 and TRPA1 receptors contributes to an enhanced excitability of sensory C-fibers.

Allergic asthma is an inflammatory disease that is associated with airway hyperreactivity (AHR). AHR is characterized by cough, dyspnea and an enhanced sputum production in response to many environmental stimuli such as cold air, cigarette smoke and ozone. Evidence has been provided that sensory neurons, especially the unmyelinated C-fibers, might contribute to AHR. Airway innervating C-fibers express transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1). TRPV1 and TRPA1 are cation channels that can be activated by most of those irritants which cause AHR-related asthma attacks. Therefore, it is speculated that TRP channels are key players in the pathogenesis of AHR. However, the mechanisms by which TRPV1 and TRPA1 are regulated in asthma are unclear. At least 22 fibroblast growth factors (FGFs) have been identified which activate FGF receptors. FGFs are located in epithelial and endothelial basement membranes, and are upregulated in the airways in allergic asthma. The aim of this study is to investigate if FGFs can regulate TRPV1 and TRPA1 sensitivity.

AIMS:In patients with bladder outlet obstruction (BOO), dysregulation of bladder afferent neurons seems to contribute to irritative symptoms. Cholinergic receptors, addressed by both neuronal and non-neuronal (urothelial) acetylcholine, can alter neuronal excitability. Thus we investigated the influence of BOO on the expression of muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors in the lumbosacral dorsal root ganglia (DRG) of mice. MAIN METHODS:BOO was induced in 13 C57/BL6 mice by partial suturing of the urethra. Eleven mice were sham-operated (loose/freely movable suture around the urethra), and eleven untreated mice served as controls. Cystometry was performed five weeks later in conscious mice. DRG at segmental levels L5-S2 were dissected and real-time quantitative PCR was performed. Expression of mAChR subtypes M1-M5 and nAChR subunits α2-7, α9-10, β2-4 was examined. KEY FINDINGS:Expression of all mAChR subtypes and nAChR subunits α3-7, α10, β2-4 was detected. Expression of α2 and α9 was absent. Rank order of expression was M2>M4>M3>M5>M1, α3≥α6>α7>α4>α10>α5 and β2>β4>β3 in untreated animals. BOO mice presented distinct obstruction with development of residual urine. Sham mice showed only minimal BOO. Relative mRNA expression of nAChR subunits revealed significant reduction of α3, α5, α6, α10 and β4 in sham-operated vs. untreated mice. In BOO vs. sham-operated mice, reduction of nAChR subunits α10 (p=0.038) and α5 (p=0.053) was found. SIGNIFICANCE:BOO has a considerable impact on nAChR, but not on mAChR mRNA expression in sensory neurons. We hypothesize that a reduction in mRNA expression of nAChR subunits represents a link to altered sensitivity under non-obstructive conditions.