Liposomes Coated with Novel Synthetic Bifunctional Chitosan Derivatives as Potential Carriers of Anticancer Drugs.

In this study, liposomes coated with novel multifunctional polymers were proposed as an innovative platform for tumor targeted drug delivery. Novel Folic acid-Cysteine-Thiolated chitosan (FTC) derivatives possessing active targeting ability and redox responsivity were synthesized, characterized, and employed to develop FTC-coated liposomes. Liposomes were characterized for size, surface charge and drug encapsulation efficiency before and after coating. The formation of a coating layer on liposomal surface was confirmed by the slight increase in particle size and by zeta-potential changes. FTC-coated liposomes showed a redox-dependent drug release profile: good stability at physiological conditions and rapid release of liposome-entrapped calcein in presence of glutathione. Moreover, the uptake and cytotoxic activity of doxorubicin-loaded FTC-coated liposomes was evaluated on murine B16-F10 and human SKMEL2 melanoma cancer cells. Results demonstrated enhanced uptake and antitumor efficacy of FTC-coated liposomes compared to control chitosan-coated liposomes in both cancer lines, which is attributed to higher cellular uptake via folate receptor-mediated endocytosis and to triggered drug release by the reductive microenvironment of tumor cells. The proposed novel liposomes show great potential as nanocarriers for targeted therapy of cancer.

Development and Evaluation of Biodegradable Core-Shell Microfibrous and Nanofibrous Scaffolds for Tissue Engineering Applications.

Tissue engineering scaffolds as three-dimensional substrates may serve as ideal templates for tissue regeneration by simulating the structure of the extracellular matrix (ECM). Many biodegradable synthetic polymers, either hydrophobic, like Poly-ε-caprolactone (PCL), or hydrophilic, like Poly(Vinyl Alcohol) (PVA), are widely used as candidate bioactive materials for fabricating tissue engineering scaffolds. However, a combination of good cytocompatibility of hydrophilic polymers with good biomechanical performance of hydrophobic polymers could be beneficial for the in vivo performance of the scaffolds. In this study, we aimed to fabricate biodegradable fibrous scaffolds by combining the properties of hydrophobic PCL with those of hydrophilic PVA and evaluate their properties in comparison with pristine PCL scaffolds. Therefore, single-layered PCL scaffolds, sequential tri-layered (PVA/PCL/PVA), and core-shell (PVA as shell and PCL as core) composite scaffolds were developed utilizing the electrospinning technique. The material structural and biomechanical properties of the electrospun scaffolds, before and after their hydrolytic degradation over a seven-month period following storage in phosphate-buffered saline (PBS) at 37 °C, were comprehensively compared. In addition, human embryonic kidney cells (HEK-293) were cultured on the scaffolds to investigate potential cell attachment, infiltration, and proliferation. The results demonstrated the long-term efficacy of core-shell biodegradable fibrous scaffolds in comparison to single-layers PCL and tri-layers PVA/PCL/PVA, not only due to its superior morphological characteristics and mechanical properties, but also due to its ability to promote homogeneous cell distribution and proliferation, without any external chemical or physical stimuli.

Murine Dermal Lymphatic Endothelial Cell Isolation.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes. Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Genetic deletion or tyrosine phosphatase inhibition of PTPRZ1 activates c-Met to up-regulate angiogenesis and lung adenocarcinoma growth.

Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) expressed in endothelial cells and required for stimulation of cell migration by vascular endothelial growth factor A165 (VEGFA165 ) and pleiotrophin (PTN). It is also over or under-expressed in various tumor types. In this study, we used genetically engineered Ptprz1-/- and Ptprz1+/+ mice to study mechanistic aspects of PTPRZ1 involvement in angiogenesis and investigate its role in lung adenocarcinoma (LUAD) growth. Ptprz1-/- lung microvascular endothelial cells (LMVEC) have increased angiogenic features compared with Ptprz1+/+ LMVEC, in line with the increased lung angiogenesis and the enhanced chemically induced LUAD growth in Ptprz1-/- compared with Ptprz1+/+ mice. In LUAD cells isolated from the lungs of urethane-treated mice, PTPRZ1 TP inhibition also enhanced proliferation and migration. Expression of beta 3 (ß3 ) integrin is decreased in Ptprz1-/- LMVEC, linked to enhanced VEGF receptor 2 (VEGFR2), c-Met tyrosine kinase (TK) and Akt kinase activities. However, only c-Met and Akt seem responsible for the enhanced endothelial cell activation in vitro and LUAD growth and angiogenesis in vivo in Ptprz1-/- mice. A selective PTPRZ1 TP inhibitor, VEGFA165 and PTN also activate c-Met and Akt in a PTPRZ1-dependent manner in endothelial cells, and their stimulatory effects are abolished by the c-Met TK inhibitor (TKI) crizotinib. Altogether, our data suggest that low PTPRZ1 expression is linked to worse LUAD prognosis and response to c-Met TKIs and uncover for the first time the role of PTPRZ1 in mediating c-Met activation by VEGFA and PTN.

Non-Oncogene Addiction of KRAS-Mutant Cancers to IL-1ß via Versican and Mononuclear IKKß.

Kirsten rat sarcoma virus (KRAS)-mutant cancers are frequent, metastatic, lethal, and largely undruggable. While interleukin (IL)-1ß and nuclear factor (NF)-κB inhibition hold promise against cancer, untargeted treatments are not effective. Here, we show that human KRAS-mutant cancers are addicted to IL-1ß via inflammatory versican signaling to macrophage inhibitor of NF-κB kinase (IKK) ß. Human pan-cancer and experimental NF-κB reporter, transcriptome, and proteome screens reveal that KRAS-mutant tumors trigger macrophage IKKß activation and IL-1ß release via secretory versican. Tumor-specific versican silencing and macrophage-restricted IKKß deletion prevents myeloid NF-κB activation and metastasis. Versican and IKKß are mutually addicted and/or overexpressed in human cancers and possess diagnostic and prognostic power. Non-oncogene KRAS/IL-1ß addiction is abolished by IL-1ß and TLR1/2 inhibition, indicating cardinal and actionable roles for versican and IKKß in metastasis.

Development and Comparative In Vitro and In Vivo Study of BNN27 Mucoadhesive Liposomes and Nanoemulsions for Nose-to-Brain Delivery.

Intranasal administration offers an alternative and promising approach for direct nose-to-brain delivery. Herein, we developed two chitosan (CHT)-coated (and uncoated) nanoformulations of BNN27 (a synthetic C-17-spiro-dehydroepiandrosterone analogue), liposomes (LIPs), and nanoemulsions (NEs), and compared their properties and brain disposition (in vitro and in vivo). LIPs were formulated by thin film hydration and coated with CHT by dropwise addition. BNN27-loaded NEs (BNEs) were developed by spontaneous emulsification and optimized for stability and mucoadhesive properties. Mucoadhesive properties were evaluated by mucin adherence. Negatively charged CHT-coated LIPs (with 0.1% CHT/lipid) demonstrated the highest coating efficiency and mucoadhesion. BNEs containing 10% w/w Capmul-MCM and 0.3% w/w CHT demonstrated the optimal properties. Transport of LIP or NE-associated rhodamine-lipid across the blood-brain barrier (in vitro) was significantly higher for NEs compared to LIPs, and the CHT coating demonstrated a negative effect on transport. However, the CHT-coated BNEs demonstrated higher and faster in vivo brain disposition following intranasal administration compared to CHT-LIPs. For both BNEs and LIPs, CHT-coating resulted in the increased (in vivo) brain disposition of BNN27. Current results prove that CHT-coated NEs consisting of compatible nasal administration ingredients succeeded in to delivering more BNN27 to the brain (and faster) compared to the CHT-coated LIPs.

KRAS signaling in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to death. MPM harbors loss-of-function mutations in BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here, we show that a proportion of human MPM harbor point mutations, copy number alterations, and overexpression of KRAS with or without TP53 changes. These are likely pathogenic, since ectopic expression of mutant KRASG12D in the pleural mesothelium of conditional mice causes epithelioid MPM and cooperates with TP53 deletion to drive a more aggressive disease form with biphasic features and pleural effusions. Murine MPM cell lines derived from these tumors carry the initiating KRASG12D lesions, secondary Bap1 alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS alterations alone or in accomplice with TP53 alterations likely play an important and underestimated role in a proportion of patients with MPM, which warrants further exploration.

Haematologic Markers and Tonsil-to-Body Weight Ratio to Assist Adenotonsillar Hypertrophy Diagnosis.

Aims Diagnosis of Adenotonsillar Hypertrophy (ATH), the leading cause of pediatric Obstructive Sleep Apnea (OSA), depends on physical exam via Brodsky's staging of tonsils. This study investigates the associations of ATH with patient parameters, and balances in-office tonsil hypertrophy appraisal against true organ mass. Materials and Methods A prospective cohort was formed of 103 children operated for ATH, and 31 matched controls. Demographic, clinical and tympanographic data, as well as Complete Blood Count (CBC) indices were compared. Absolute and relative to total body weight tonsil specimen mass were correlated with Brodsky's score. Results Tonsillar size indices were significantly raised in ATH patients. Elevated leukocytes (P = 0.012) and increased neutrophil percentage (P = 0.025) conveyed higher ATH risk. Subjective evaluation of tonsils graded 1 or 2 correlated significantly with absolute (P = 0.001) and relative (P = 0.006) objective measurements. Brodsky's score 3 and 4 displayed marginal significant association with relative (P = 0.050) but not with true (P = 0.989) mass. Conclusion An occult hematologic inflammatory response was detected in ATH children. Clinical estimation of severely hypertrophic tonsils should be adjusted for total body weight. Trial Registration Number: NCT03541434 (clinicaltrials.gov).

Synergistic effect of cold atmospheric pressure plasma and free or liposomal doxorubicin on melanoma cells.

The aim of the present study was to investigate combined effects of cold atmospheric plasma (CAP) and the chemotherapeutic drug doxorubicin (DOX) on murine and human melanoma cells, and normal cells. In addition to free drug, the combination of CAP with a liposomal drug (DOX-LIP) was also studied for the first time. Thiazolyl blue tetrazolium bromide (MTT) and Trypan Blue exclusion assays were used to evaluate cell viability; the mechanism of cell death was evaluated by flow cytometry. Combined treatment effects on the clonogenic capability of melanoma cells, was also tested with soft agar colony formation assay. Furthermore the effect of CAP on the cellular uptake of DOX or DOX-LIP was examined. Results showed a strong synergistic effect of CAP and DOX or DOX-LIP on selectively decreasing cell viability of melanoma cells. CAP accelerated the apoptotic effect of DOX (or DOX-LIP) and dramatically reduced the aggressiveness of melanoma cells, as the combination treatment significantly decreased their anchorage independent growth. Moreover, CAP did not result in increased cellular uptake of DOX under the present experimental conditions. In conclusion, CAP facilitates DOX cytotoxic effects on melanoma cells, and affects their metastatic potential by reducing their clonogenicity, as shown for the first time.

Engineered versus hybrid cellular vesicles as efficient drug delivery systems: a comparative study with brain targeted vesicles.

Herein we elaborated on methods to load cellular vesicles (CVs) and to incorporate cholesterol (Chol) and PEG lipids in their membrane, for enhancing the potential of such engineered CVs (e-CVs) as drug carriers. Hybrids formed by fusion between PEGylated liposomes (PEG-LIP) and CVs were evaluated as alternatives to e-CV, for the first time. Freeze-thawing cycles (FT) and incubation protocols were tested, and vesicle fusion was monitored by FRET dilution. B16F10, hCMEC/D3, and LLC cells were used for e-CV or hybrid development, and FITC-dextran as a model hydrophilic drug. Results show that dehydration rehydration vesicle (DRV) method is optimal for highest CV loading and integrity, while optimal protocols for Chol/PEG enrichment were identified. FT was found to be more efficient than incubation for hybrid formation. Interestingly, despite their high Chol content, CVs had very low integrity that was not increased by enrichment with Chol, but only after PEG coating; e-CVs demonstrated higher integrity than hybrids. Vesicle uptake by hCMEC cells is in the order: LIP < e-CVs < Hybrids ≤ CVs (verified by confocal microscopy); the higher PEG content of e-CVs is possibly the reason for their reduced cell uptake. While CV and hybrid uptake are highly caveolin-dependent, e-CVs mostly follow clathrin-dependent pathways. In vivo and ex vivo results show that brain accumulation of hybrids is only slightly higher that of CVs, indicating that the surface PEG content of hybrids is not sufficient to prevent uptake by macrophages of the reticuloendothelial system. Taking together with the fact that subjection of CVs to FT cycles reduced their cellular uptake, it is concluded that PEGylated e-CVs are better than hybrids as brain-targeted drug carriers.

Liposomes Decorated with 2-(4'-Aminophenyl)benzothiazole Effectively Inhibit Aß1-42 Fibril Formation and Exhibit in Vitro Brain-Targeting Potential.

The potential of 2-benzothiazolyl-decorated liposomes as theragnostic systems for Alzheimer's disease was evaluated in vitro, using PEGylated liposomes that were decorated with two types of 2-benzothiazoles: (i) the unsubstituted 2-benzothiazole (BTH) and (ii) the 2-(4-aminophenyl)benzothiazole (AP-BTH). The lipid derivatives of both BTH-lipid and AP-BTH-lipid were synthesized, for insertion in liposome membranes. Liposomes (LIP) containing three different concentrations of benzothiazoles (5, 10, and 20%) were formulated, and their stability, integrity in the presence of serum proteins, and their ability to inhibit ß-amyloid (1-42) (Αß42) peptide aggregation (by circular dichroism (CD) and thioflavin T (ThT) assay), were evaluated. Additionally, the interaction of some LIP with an in vitro model of the blood-brain barrier (BBB) was studied. All liposome types ranged between 92 and 105 nm, with the exception of the 20% AP-BTH-LIP that were larger (180 nm). The 5 and 10% AP-BTH-LIP were stable when stored at 4 °C for 40 days and demonstrated high integrity in the presence of serum proteins for 7 days at 37 °C. Interestingly, CD experiments revealed that the AP-BTH-LIP substantially interacted with Αß42 peptides and inhibited fibril formation, as verified by ThT assay, in contrast with the BTH-LIP, which had no effect. The 5 and 10% AP-BTH-LIP were the most effective in inhibiting Αß42 fibril formation. Surprisingly, the AP-BTH-LIP, especially the 5% ones, demonstrated high interaction with brain endothelial cells and high capability to be transported across the BBB model. Taken together, the current results reveal that the 5% AP-BTH-LIP are of high interest as novel targeted theragnostic systems against AD, justifying further in vitro and in vivo exploitation.

Osteopontin drives KRAS-mutant lung adenocarcinoma.

Increased expression of osteopontin (secreted phosphoprotein 1, SPP1) is associated with aggressive human lung adenocarcinoma (LADC), but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced LADC. We combined mouse models of tobacco carcinogen-induced LADC, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human LADC and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller LADC with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target.

Interleukin-1ß provided by KIT-competent mast cells is required for KRAS-mutant lung adenocarcinoma.

Mast cells (MC) have been identified in human lung adenocarcinoma (LADC) tissues, but their functional role has not been investigated in vivo. For this, we applied three mouse models of KRAS-mutant LADC to two different MC-deficient mouse strains (cKitWsh and Cpa3.Cre). Moreover, we derived MC gene signatures from murine bone marrow-derived MC and used them to interrogate five human cohorts of LADC patients. Tumor-free cKitWsh and Cpa3.Cre mice were deficient in alveolar and skin KIT-dependent (KIT+) MC, but cKitWsh mice retained normal KIT-independent (KIT-) MC in the airways. Both KIT+ and KIT- MC infiltrated murine LADC to varying degrees, but KIT+ MC were more abundant and promoted LADC initiation and progression through interleukin-1ß secretion. KIT+ MC and their transcriptional signature were significantly enriched in human LADC compared to adjacent normal tissue, especially in the subset of patients with KRAS mutations. Importantly, MC density increased with tumor stage and high overall expression of the KIT+ MC signature portended poor survival. Collectively, our results indicate that KIT+ MC foster LADC development and represent marked therapeutic targets.

Club cells form lung adenocarcinomas and maintain the alveoli of adult mice.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease.

Tobacco chemical-induced mouse lung adenocarcinoma cell lines pin the prolactin orthologue proliferin as a lung tumour promoter.

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers' LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease.

Exosomes and Exosome-Inspired Vesicles for Targeted Drug Delivery.

The similarities between exosomes and liposomes, together with the high organotropism of several types of exosomes, have recently prompted the development of engineered-exosomes or exosome-mimetics, which may be artificial (liposomal) or cell-derived vesicles, as advanced platforms for targeted drug delivery. Here, we provide the current state-of-the-art of using exosome or exosome-inspired systems for drug delivery. We review the various approaches investigated and the shortcomings of each approach. Finally the challenges which have been identified to date in this field are summarized.

IκB Kinase α Is Required for Development and Progression of KRAS-Mutant Lung Adenocarcinoma.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB-activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKß in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D Using NFκB reporter mice and conditional deletions of IKKα and IKKß, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBß, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKß inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939-51. ©2018 AACR.

Myeloid-derived interleukin-1ß drives oncogenic KRAS-NF-κΒ addiction in malignant pleural effusion.

Malignant pleural effusion (MPE) is a frequent metastatic manifestation of human cancers. While we previously identified KRAS mutations as molecular culprits of MPE formation, the underlying mechanism remained unknown. Here, we determine that non-canonical IKKα-RelB pathway activation of KRAS-mutant tumor cells mediates MPE development and this is fueled by host-provided interleukin IL-1ß. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1ß fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1ß-mediated NF-κB induction in KRAS-mutant tumor cells, as well as their resulting MPE-competence, can only be blocked by co-inhibition of both KRAS and IKKα, a strategy that overcomes drug resistance to individual treatments. Hence we show that mutant KRAS facilitates IKKα-mediated responsiveness of tumor cells to host IL-1ß, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance.

RAS oncogenes direct metastasis.

RAS genes are cardinal driver oncogenes frequently and differentially mutated across bodily tumors. Their tumorigenic potential has been mainly ascribed to autonomous promotion of tumor cell proliferation and survival. However, recent evidence shows that RAS oncogenes also function to define metastatic tropism. Interestingly, RAS-driven metastasis is mediated by distinct chemokine sets that signal to endothelial and myeloid cells.

Mutant KRAS promotes malignant pleural effusion formation.

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.