Lung Tumor Cells Research Articles

The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

Dual Inhibition of MEK and AXL Targets Tumor Cell Heterogeneity and Prevents Resistant Outgrowth Mediated by the Epithelial-to-Mesenchymal Transition in NSCLC.

The epithelial-to-mesenchymal transition (EMT) is a dynamic epigenetic reprogramming event that occurs in a subset of tumor cells and is an initiating step toward invasion and distant metastasis. The process is reversible and gives plasticity to cancer cells to survive under variable conditions, with the acquisition of cancer stem cell-like characteristics and features such as drug resistance. Therefore, understanding survival dependencies of cells along the phenotypic spectrum of EMT will provide better strategies to target the spatial and temporal heterogeneity of tumors and prevent their ability to bypass single-inhibitor treatment strategies. To address this, we integrated the data from a selective drug screen in epithelial and mesenchymal KRAS/p53 (KP)-mutant lung tumor cells with separate datasets including reverse-phase protein array and an in vivo shRNA dropout screen. These orthogonal approaches identified AXL and MEK as potential mesenchymal and epithelial cell survival dependencies, respectively. To capture the dynamicity of EMT, incorporation of a dual fluorescence EMT sensor system into murine KP lung cancer models enabled real-time analysis of the epigenetic state of tumor cells and assessment of the efficacy of single agent or combination treatment with AXL and MEK inhibitors. Both two- and three-dimensional culture systems and in vivo models revealed that this combination treatment strategy of MEK plus AXL inhibition synergistically killed lung cancer cells by specifically targeting each phenotypic subpopulation. In conclusion, these results indicate that cotargeting the specific vulnerabilities of EMT subpopulations can prevent EMT-mediated drug resistance, effectively controlling tumor cell growth and metastasis. SIGNIFICANCE: This study shows that a novel combination of MEK and AXL inhibitors effectively bypasses EMT-mediated drug resistance in KRAS/p53-mutant non-small cell lung cancer by targeting EMT subpopulations, thereby preventing tumor cell survival.

Cortisol promotes breast-to-brain metastasis through the blood-cerebrospinal fluid barrier.

BackgroundElevated basal cortisol levels are present in women with primary and metastatic breast cancer. Although cortisol's potential role in breast‐to‐brain metastasis has yet to be sufficiently studied, prior evidence indicates that it functions as a double‐edged sword—cortisol induces breast cancer metastasis in vivo, but strengthens the blood‐brain‐barrier (BBB) to protect the brain from microbes and peripheral immune cells.AimsIn this study, we provide a novel examination on whether cortisol's role in tumor invasiveness eclipses its supporting role in strengthening the CNS barriers. We expanded our study to include the blood‐cerebrospinal fluid barrier (BCSFB), an underexamined site of tumor entry.Methods and ResultsUtilizing in vitro BBB and BCSFB models to measure barrier strength in the presence of hydrocortisone (HC). We established that lung tumor cells migrate through both CNS barriers equally while breast tumors cells preferentially migrate through the BCSFB. Furthermore, HC treatment increased breast‐to‐brain metastases (BBM) but not primary breast tumor migratory capacity. When examining the transmigration of breast cancer cells across the BCSFB, we demonstrate that HC induces increased traversal of BBM but not primary breast cancer. We provide evidence that HC increases tightness of the BCSFB akin to the BBB by upregulating claudin‐5, a tight junction protein formerly acknowledged as exclusive to the BBB.ConclusionOur findings indicate, for the first time that increased cortisol levels facilitate breast‐to‐brain metastasis through the BCSFB—a vulnerable point of entry which has been typically overlooked in brain metastasis. Our study suggests cortisol plays a pro‐metastatic role in breast‐to‐brain metastasis and thus caution is needed when using glucocorticoids to treat breast cancer patients.

Non-Thermal Intervention of Lung Tumor by Core-Shell Magnetic Nanoparticles in a Magnetic Field

K-Ras mutations result in normal cells dividing uncontrollably and becoming cancerous. The prognosis is currently poor for patients due to the lack of drugs that can effectively target these mutations. In this study, magnetic nanoparticles (MNPs) were prepared, characterized, and cooperated with a magnetic field to intervene in the growth of lung tumor cells. The rise in temperature of a stimulation coil was studied by numerical calculation. The non-thermal effects of MNPs under a magnetic force were analyzed. The cell experiments showed that the growth of A549 tumor cells slowed down. The result of a wound-healing assay also indicated that the migration of tumor cells was suppressed. Compared with magnetic stimulation without MNPs, MNPs enhanced the inhibitory effects of a magnetic field. This study suggests a new way to treat K-Ras driven lung tumors using non-thermal effects of MNPs without the side effects caused by thermal effects.

Mesenchymal stromal cell delivery of oncolytic immunotherapy improves CAR-T cell antitumor activity

The immunosuppressive tumor microenvironment (TME) is a formidable barrier to the success of adoptive cell therapies for solid tumors. Oncolytic immunotherapy with engineered adenoviruses (OAd) may disrupt the TME by infecting tumor cells, as well as surrounding stroma, to improve the functionality of tumor-directed chimeric antigen receptor (CAR)-T cells, yet efficient delivery of OAds to solid tumors has been challenging. Here we describe how mesenchymal stromal cells (MSCs) can be used to systemically deliver a binary vector containing an OAd together with a helper-dependent Ad (HDAd; combinatorial Ad vector [CAd]) that expresses interleukin-12 (IL-12) and checkpoint PD-L1 (programmed death-ligand 1) blocker. CAd-infected MSCs deliver and produce functional virus to infect and lyse lung tumor cells while stimulating CAR-T cell anti-tumor activity by release of IL-12 and PD-L1 blocker. The combination of this approach with administration of HER.2-specific CAR-T cells eliminates 3D tumor spheroids invitro and suppresses tumor growth in two orthotopic lung cancer models invivo. Treatment with CAd MSCs increases the overall numbers of human Tcells invivo compared to CAR-T cell only treatment and enhances their polyfunctional cytokine secretion. These studies combine the predictable targeting of CAR-T cells with the advantages of cancer cell lysis and TME disruption by systemic MSC delivery of oncolytic virotherapy: incorporation of immunostimulation by cytokine and checkpoint inhibitor production through the HDAd further enhances anti-tumor activity.

Single-cell genetic analysis of lung tumor cells based on self-driving micro-cavity array chip

Lung cancer is one of the common malignant tumors with a high incidence and mortality rate. Targeted therapies are efficient on lung cancer patients with specific gene mutations. Circulating tumor cells (CTCs) are used for liquid biopsy, providing genetic information for lung cancer treatment selection and prognosis. We developed a less costly self-driving micro-cavity array for simple molecular analysis at a single cell level to examine the genetic make-up of CTCs. This chip integrated sample detection structure and vacuum driving system to achieve cell loading, lysing, isothermal amplification (LAMP), and signal read-out on one chip. We used the “film-polydimethylsiloxane (PDMS) chip-film” structure and oil sealing method during amplification reaction to minimize water loss. We then conducted a LAMP assay using the self-driving device to detect epidermal growth factor receptor (EGFR) L858R mutation and identified an excellent linear in the range between 101-104 copies/μL (R2 = 0.997). We finally assessed the EGFR L858R gene expression of lung tumor cells (H1975 cells) as putative CTCs using the proposed detection platform. We discovered its ability to perform genetic analysis at the single-cell level. The EGFR L858R mutational gene expression levels were different in H1975 cells. In conclusion, the self-driving micro-cavity array is a less costly and simple tool for mutational gene profiling of single lung CTC. Besides, it can be used in personalized therapy and efficacy monitoring.

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

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

Overcoming hypoxia-induced resistance of pancreatic and lung tumor cells by disrupting the PERK-NRF2-HIF-axis

Hypoxia-induced resistance of tumor cells to therapeutic treatment is an unresolved limitation due to poor vascular accessibility and protective cell adaptations provided by a network, including PERK, NRF2, and HIF signaling. All three pathways have been shown to influence each other, but a detailed picture remains elusive. To explore this crosstalk in the context of tumor therapy, we generated human cancer cell lines of pancreatic and lung origin carrying an inducible shRNA against NRF2 and PERK. We report that PERK-related phosphorylation of NRF2 is only critical in Keap1 wildtype cells to escape its degradation, but shows no direct effect on nuclear import or transcriptional activity of NRF2. We could further show that NRF2 is paramount for proliferation, ROS elimination, and radioprotection under constant hypoxia (1% O2), but is dispensable under normoxic conditions or after reoxygenation. Depletion of NRF2 does not affect apoptosis, cell cycle progression and proliferation factors AKT and c-Myc, but eliminates cellular HIF-1α signaling. Co-IP experiments revealed a protein interaction between NRF2 and HIF-1α and strongly suggest NRF2 as one of the cellular key factor for the HIF pathway. Together these data provide new insights on the complex role of the PERK-NRF2-HIF-axis for cancer growth.

Exploring the binding and cleavage activities of nickelII complexes towards DNA and proteins

Three novel nickel(ii) complexes with cis octahedral geometry display excellent binding and cleavage affinity towards DNA and proteins. Furthermore, all complexes show superior cytotoxicity against human lung (A549) and breast (MCF-7) tumor cells.

Cell-cell interactions via non-covalent click chemistry.

Metabolic glycoengineering with unnatural sugars became a valuable tool for introducing recognition markers on the cell membranes via bioorthogonal chemistry. By using this strategy, we functionalized the surface of tumor and T cells using complementary artificial markers based on both β-cyclodextrins (β-CDs) and adamantyl trimers, respectively. Once tied on cell surfaces, the artificial markers induced cell–cell adhesion through non-covalent click chemistry. These unnatural interactions between A459 lung tumor cells and Jurkat T cells triggered the activation of natural killer (NK) cells thanks to the increased production of interleukin-2 (IL-2) in the vicinity of cancer cells, leading ultimately to their cytolysis. The ready-to-use surface markers designed in this study can be easily inserted on the membrane of a wide range of cells previously submitted to metabolic glycoengineering, thereby offering a simple way to investigate and manipulate intercellular interactions.

IL-9 Producing Tumor-Infiltrating Lymphocytes (Til) and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer

Although lung cancer is the leading cause of cancer death worldwide, the mechanisms how lung cancer cells evade the immune system remain incompletely understood. Here, we discovered IL-9-dependent signaling mechanisms that drive immune evasion in non-small cell lung cancer (NSCLC). We found increased IL-9 and IL-21 production by T cells in the tumoral region of the lung of patients with NSCLC, suggesting the presence of Th9 cells in the lung tumor microenvironment. Moreover, we noted IL-9 producing Tregs in NSCLC. IL-9 target cells in NSCLC consisted of IL-9R+ tumor cells and tumor-infiltrating lymphocytes. In two murine experimental models of NSCLC, and in vitro , IL-9 prevented cell death and controlled growth of lung adenocarcinoma cells. Targeted deletion of IL-9 resulted in successful lung tumor rejection in vivo associated with an induction of IL-21 and reduction of lung pSTAT3, Th17 and Treg cells. Finally, anti-IL-9 antibody immunotherapy resulted in suppression of tumor development even in established experimental NSCLC and was associated with reduced IL-10 production and induction of IL-21 in the lung. In conclusion, our findings indicate that IL-9 drives immune escape of lung tumor cells via effects on tumor cell survival and tumor infiltrating T cells. Thus, strategies blocking IL-9 emerge as a new approach for clinical therapy of lung cancer. Funding: This work was supported by DFG grant FI817-5-1,2 and by a DFG FI817- 5-3 granted to SF. M.T.C. work is supported by a DFG CH 1428/2-1. MHK was supported by NIH grant AI057459. Declaration of Interest: None to declare. Ethical Approval: Our human study was performed at the Friedrich-Alexander-University Erlangen-Nurnberg, Germany, after being approved by the ethics review board of the University of Erlangen (Re-No: 56_12B; DRKS-ID: DRK5376).

Surface peptide functionalization of zeolitic imidazolate framework-8 for autonomous homing and enhanced delivery of chemotherapeutic agent to lung tumor cells.

Chemotherapeutic agents used in treating certain cancer types operate in a non-selective manner tending to accumulate in normal, healthy tissue when high doses are used. To mitigate the toxicity effect resulting from this, there is an urgent need to develop active nano delivery systems capable of regulating optimal doses specifically to cancer cells without harming adjacent normal cells. Herein, we report a versatile nanoparticle - zeolitic imidazolate framework-8 (nZIF-8) - that is loaded with a chemotherapeutic agent (gemcitabine; GEM) and surface-functionalized with an autonomous homing system (Arg-Gly-Asp peptide ligand; RGD) via a straightforward, one-pot solvothermal reaction. Successful functionalization of the surface of nZIF-8 loaded GEM (GEM⊂nZIF-8) with RGD was proven by spectroscopic and electron microscopy techniques. This surface-functionalized nanoparticle (GEM⊂RGD@nZIF-8) exhibited enhanced uptake in human lung cancer cells (A549), compared with non-functionalized GEM⊂nZIF-8. The GEM⊂RGD@nZIF-8, experienced not only efficient uptake within A549, but also induced obvious cytotoxicity (75% at a concentration of 10 μg mL-1) and apoptosis (62%) after 48 h treatment when compared to the nanoparticle absent of the RGD homing system (GEM⊂nZIF-8). Most importantly, this surface-functionalized nanoparticle was more selective towards lung cancer cells (A549) than normal human lung fibroblast cells (MRC-5) with a selectivity index (SI) of 3.98. This work demonstrates a new one-pot strategy for realizing a surface-functionalized zeolitic imidazolate framework that actively targets cancer cells via an autonomous homing peptide system to deliver a chemotherapeutic payload effectively.

Liver cell membrane antibody, kidney injury molecule and lungs tumour antigen expression on the MCF-7 cell induced breast cancer administered with Citrus limon juice and tamoxifen in Sprawgue dawley rats

Monitoring and evaluation have been one of the major measures to determine possible progression of response of disorder to the alternative treatments, however Anti-Liver Cell membrane Antibody, Kidney Injury Molecule and Lungs Tumour Antigen Expression on the MCF-7 Cell Induced Breast Cancer Rats administered with Citrus Limon juice and Tamoxifen were carried out, Over one hundred and twenty Sprague dawley rats of 40 days old average body weight 180-220g were divided into ten (10) containing of 12 animals per group, group 1 was control, fed only with rat chow and water, group 2 was MCF-7 cell line induced rats alone (BCIR only), group 3 Citrus Limon juice (CLJ) at 8.88%, Group 4 Citrus Limon juice (CLJ) at 17.32%, group 5 Citrus Limon juice (CLJ) at 25.98%, group 6 was given 0.2mg/kg of Tamoxifen alone, group 7 (BCIR+CLJ at 8.88%), group 8 (BCIR+CLJ at 17.32%), group 9 (BCIR+CLJ at 25.98%) and group (BCIR+ 0.2mg/kg of Tamoxifen). Acute and sub – acute toxicity were carried out after the establishment of safety dose following determination of LD50, on the fresh Citrus Limon juice Breast cancer induction and Tumor sizes were analysed. At the end of the administration, animals were sacrificed, fresh blood was taken, centrifuged at 3000 rpm, serum was collected and stored at 8oc Breast Cancer Tumor Associated Assays, Kidney Injury, Liver cell Membrane Antibody and Lung Tumor Antigens were analysed. Result, Mammary tumor induction was succeeded in all the MCF-7 cell line induced groups with minimum number of morbidity. Tumor size was significantly increase in tumor mass BCIR only at P>0.001 when compared to BCIR + CLJ and BCIR + Tamoxifen groups. Immunoogical expression of Breast Cancer Associated Tumor revealed significant increase suppressor gene/protein, Immunocytochemical in-vivo of Breast Cancer Associated Organs showed significant increase in KIM , LCMA and LTA at P>0.001 in BCIR when compared to all the BCIR + CLJ, BCIR + Tamoxifen and other non-BCIR groups. In conclusion, this study showed that KIM , LCMA and LTA exhibited strong Immunochemical expression of Citrus Limon juice potency possessing possible anticancer activities without posing pathological conditions on the body during and after usage, hence KIM, LCMA and LTA are demonstrated to be valuable in the monitoring and evaluation progression of tumor as Citrus Limon Juice could be used as alternative therapy in the treatment of Breast cancer that are hormonal dependant in similar manner to Tamoxifen.

Inhibition of the FGF/FGFR System Induces Apoptosis in Lung Cancer Cells via c-Myc Downregulation and Oxidative Stress.

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.

Synthesis and evaluation of novel fluorinated hematoporphyrin ether derivatives for photodynamic therapy

A photosensitizer with high phototoxicity, suitable amphipathy and low dark toxicity could play a pivotal role in photodynamic therapy (PDT). In this study, a facile and versatile approach was adopted to synthesize a series of novel fluorinated hematoporphyrin ether derivatives (I1-I5 and II1-II4), and the photodynamic activities of these compounds were studied. Compared to hematoporphyrin monomethyl ether (HMME), all PSs showed preferable photodynamic activity against A549 lung tumor cells. The longest visible absorption wavelength of these compounds was approximately 622 nm. Among them, II3 revealed the highest singlet oxygen yield (0.0957 min−1), the strongest phototoxicity (IC50 = 1.24 μM), the lowest dark toxicity in vitro, and exhibited excellent anti-tumor effects in vivo. So compound II3 could act as new drug candidate for photodynamic therapy.

Extracellular AGR2 triggers lung tumour cell proliferation through repression of p21CIP1

The human Anterior GRadient 2 (AGR2) protein is an Endoplasmic Reticulum (ER)-resident protein which belongs to the Protein-Disulfide Isomerase (PDI) superfamily and is involved to productive protein folding in the ER. As such AGR2, often found overexpressed in adenocarcinomas, contributes to tumour development by enhancing ER proteostasis. We previously demonstrated that AGR2 is secreted (extracellular AGR2 (eAGR2)) in the tumour microenvironment and plays extracellular roles independent of its ER functions. Herein, we show that eAGR2 triggers cell proliferation and characterize the underlying molecular mechanisms. We demonstrate that eAGR2 enhances tumour cell growth by repressing the tumour suppressor p21CIP1. Our findings shed light on a novel mechanism through which eAGR2 behaves as a growth factor in the tumour microenvironment, independently of its ER function, thus promoting tumour cell growth through repression of p21CIP1. Our results provide a rationale for targeting eAGR2/p21CIP1-based signalling as a potential therapeutic target to impede tumour growth.

Identification of protocatechuic acid as a novel blocker of epithelial-to-mesenchymal transition in lung tumor cells.

Protocatechuic acid (PA) is widely distributed and commonly occurring natural compound that can exert antioxidant, anti-inflammatory, as well as anti-cancer effects. Epithelial-to-mesenchymal transition (EMT) is important cellular process that can control tumor invasion and metastasis. Here, we investigated whether PA can modulate the EMT process in basal and transforming growth factorβ-induced A549 and H1299 cells. We found that PA suppressed expression of mesenchymal markers (Fibronectin, Vimentin, and N-cadherin), MMP-9, MMP-2, twist, and snail but stimulated the levels of epithelial markers (E-cadherin and Occludin). In addition, PA can affect TGFβ-induced expression of both mesenchymal and epithelial markers. Moreover, PA abrogated migratory and invasive potential of tumor cells by reversing the EMT process. Furthermore, we found that PA suppressed EMT process by abrogating the activation of PI3K/Akt/mTOR signaling cascade in lung cancer cells.

Epithelial-mesenchymal transition classification of circulating tumor cells in lung and colon cancer patients: potential role in clinical practice.

BackgroundTumor cells undergoing epithelial-mesenchymal transition (EMT) display enhanced ability to enter the circulation, thereby being major source of circulating tumor cells (CTCs). In this study, we aimed to better understand the roles of CTC undergoing EMT in monitoring cancer progression.MethodsWe analyzed gene expression profiling of epithelial and mesenchymal markers in lung or colon tumor samples by mining TCGA database. We detected CTCs and classify their EMT phenotypes of 31 patients with lung or colon cancer by using a CanPatrol CTC-enrichment technique.ResultsThe bioinformatic analysis indicated that mesenchymal markers were expressed in a subset of lung tumor samples, and its high expression was associated with poor survival of lung cancer patients. However, in colon cancer, majority of tumor samples expressed hybrid epithelial/mesenchymal markers. CTC analysis with EMT classification showed that the number of CTCs with mesenchymal phenotype was high in lung cancer patients with the advanced stage. Dynamic CTC analysis in a lung cancer patient indicated that CTC with mesenchymal phenotype was effective to monitor tumor progression. In a colon cancer patient, dynamic CTC analysis indicated that CTC with hybrid epithelial/mesenchymal phenotypes was an effective biomarker to guide therapy.ConclusionsEncouraging results from this proof-of-concept study show that CTC with mesenchymal phenotype or hybrid epithelial/mesenchymal phenotypes could be a potential biomarker for monitoring tumor progression in lung or colon cancer respectively.

Overview of Lung Cancer Immunotherapy.

Anti-PD-(L)1 therapy represents a turning point in lung cancer immunotherapy, moving from previously ineffective enhancer strategies to immune checkpoints as standard first- and second-line therapies. This unprecedented success highlights the importance of mechanisms to escape immune attack, such PD-1/PD-L1 axis, and emphasize the importance to better understand the tumor immune microenvironment. Analyzing the specifics of immune response against lung tumor cells and how malignant cells progressively adapt to this pressure may help to understand which are the key aspects to guide the development of new therapeutic strategies. Here we review the past and present of clinical lung cancer immunotherapy and give a perspective for the future development based on emerging biological insights.

Ex vivo Live Cell Imaging of Nanoparticle-Cell Interactions in the Mouse Lung.

A successful clinical translation of novel nanoparticle-based cancer therapeutics requires a thorough preclinical investigation of their interaction with immune, tumor and endothelial cells as well as components of the tumor-microenvironment. Although high-resolution microscopy images of fixed tumor tissue specimens can provide valuable information in this regard, they are only static snapshots of a momentary event. Here we describe a superior alternative fluorescence microscopy approach to assess the feasibility of investigating nanoparticle-cell interactions in the mouse lung live and over time at nanometer resolution. We applied fluorescent lung tumor cells and Barium-based fluorescently labeled nanoparticles to nude mice or to CD68-EGFP transgenic mice for visualization of the monocyte-macrophage lineage. Shortly before imaging, fluorescently labeled lectin was intravenously injected for staining of the blood vessels. The lung was filled ex vivo with 1% agarose and individual lung lobes were imaged over time using a confocal microscope with Airyscan technology. Time series demonstrate that live cell imaging of lung lobes can be performed for at least 4 h post mortem. Time-lapse movies illustrate the dynamics of the nanoparticles within the pulmonary circulation and their uptake by immune cells. Moreover, the exchange of nanoparticle material between cancer cells was observed over time. Fluorescent monocytes in lungs of CD68-EGFP transgenic mice could be visualized within blood vessels in the process of interaction with tumor cells and nanoparticles. This high resolution ex vivo live cell imaging approach provides an excellent 4D tool to obtain valuable information on the behavior of tumor and immune cells at first encounter with nanoparticles and may contribute to the understanding of how nanoparticles interact with cells supporting the development of therapeutic strategies based on nanoparticulate drug delivery systems.