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Abstract Background Cell death within the tumor microenvironment (TME) plays a crucial role in controlling cancer by influencing the balance of tumor-specific immunity. Cancer-associated fibroblasts (CAFs) significantly contribute to tumor progression through paracrine mechanisms. We found that reprogramming of CAFs by apoptotic cancer cells suppresses tumor volume and lung metastasis. Here, we investigated the mechanisms by which the interaction between apoptotic lung cancer cells and CAFs hinders tumor growth. Methods Experimental methods including CCK assay, colony formation assay, immunoblotting, co-immunoprecipitation, qRT-PCR analysis, qRT-PCR array, apoptosis assay, ELISA, and immunofluorescent staining were used in this study. Additionally, CAFs were isolated from lung tumors of Kras-mutant (KrasLA1) mice and human lung adenocarcinoma samples using magnetic-activated cell sorting. Murine lung cancer cells (344SQ cells) along with various human cancer cell lines (A549, HCT116, and LoVo) were cultured. In animal study, conditioned medium (CM) derived from CAFs (undiluted or 50% diluted) with or without neutralizing anti-WISP-1 antibody was administered into syngeneic mice to study anti-tumoral effects. To confirm the paracrine role of WISP-1, recombinant WISP-1 (rWISP-1) was administered via intratumoral injection. Results We demonstrate that treatment with CM from lung CAFs exposed to apoptotic cancer cells suppresses proliferation and promotes apoptosis in lung cancer cells through STAT1 signaling. Pharmacologic inhibition of Notch1 activation or siRNA-mediated Notch1 silencing in CAFs reversed the antiproliferative and proapoptotic effects. Similarly, knockdown of Wnt-induced signaling protein 1 (WISP-1) in CAFs or neutralizing the CM with anti-WISP-1 antibodies reversed the antiproliferative and proapoptotic effects. WISP-1 signaled through integrin ανβ3-STAT1 signaling pathway to inhibit cancer cell growth and promote apoptosis. The in vivo introduction of CM derived from apoptotic 344SQ-exposed CAFs (ApoSQ-CAF CM) potently decelerated tumor growth. This effect was observed alongside the downregulation of proliferative and anti-apoptotic markers, while simultaneously boosting the activation of phosphorylated STAT1 and pro-apoptotic markers in CD326+ tumor cells within syngeneic immunocompetent mice. rWISP-1 effectively replicates the in vivo effects of ApoSQ-CAF CM. Conclusions These findings suggest that CM from apoptotic cancer cell-exposed CAFs may offer a promising therapeutic approach by lung cancer suppression.

Abstract Cancer-associated fibroblasts (CAFs) actively contribute to the formation of tumor-supportive microenvironments, thereby promoting cancer progression and impacting therapeutic outcomes. This study utilized global microRNA (miRNA) expression profiling to identify specific miRNAs responsible for reprogramming normal lung fibroblasts (LFs) into CAFs. miR-224 demonstrates increased expression in CAFs, and its levels are elevated in lung tumors compared to those in normal tissues, according to data from public databases. Overexpression of miR-224 in LFs increases the overall expression of CAF activation markers. Furthermore, LFs overexpressing miR-224 enhanced the migration and invasion of lung cancer cells via direct cell-to-cell contact in a co-culture system. In a mouse orthotopic injection model, miR-224 overexpression in LFs increased lung cancer metastasis. Using target prediction tools and subsequent 3′-UTR luciferase assay, Akirin1 was validated as a direct target gene of miR-224. In addition, LFs depleted of Akirin1 by siRNAs stimulated the migration and invasion of lung cancer cells compared to control LFs. Overall, these findings indicate that miR-224 induces CAF activation and promotes the migration and invasion of lung cancer cells by targeting Akirin1 in co-culture systems.

Abstract Background Brain metastasis is a common complication among patients with lung cancer, yet the underlying mechanisms remain unclear. In this study, we aimed to investigate the pathogenesis of brain metastasis in lung cancer. Methods We established highly colonizing metastatic lung cancer cells, A549-M2, through multiple implantations of A549 human lung cancer cells in the carotid artery of athymic nude mice. Results Compared to parental cells (M0), M2 cells demonstrated slower growth in culture plates and soft agar, as well as lower motility and higher adhesion, key characteristics of mesenchymal–epithelial transition (MET). Further analysis revealed that M2 cells exhibited decreased expression of epithelial–mesenchymal transition markers, including ZEB1 and Vimentin. M2 cells also demonstrated reduced invasiveness in co-culture systems. RNA sequencing and gene set enrichment analysis confirmed that M2 cells underwent MET. Intriguingly, depletion of Noggin, a BMP antagonist, was observed in M2 cells, and replenishment of Noggin restored suppressed migration and invasion of M2 cells. In addition, Noggin knockdown in control M0 cells promoted cell attachment and suppressed cell migration, suggesting that Noggin reduction during brain colonization causes inhibition of migration and invasion of metastatic lung cancer cells. Conclusions Our results suggest that lung cancer cells undergo MET and lose their motility and invasiveness during brain metastatic colonization, which is dependent on Noggin.

Abstract Pathological markers that can monitor the progression of gastric cancer (GC) may facilitate the diagnosis and treatment of patients with diffuse GC (DGC). To identify microRNAs (miRNAs) that can differentiate between early and advanced DGC in the gastric mucosa, miRNA expression profiling was performed using the NanoString nCounter method in human DGC tumors. Ectopic expression of miR-199a and miR-199b (miR-199a/b) in SNU601 human GC cells accelerated the growth rate, viability, and motility of cancer cells and increased the tumor volume and weight in a mouse xenograft model. To study their clinicopathological roles in patients with GC, miR-199a/b levels were measured in human GC tumor samples using in situ hybridization. High miR-199a/b expression level was associated with enhanced lymphovascular invasion, advanced T stage, and lymph-node metastasis. Using the 3′-untranslated region (UTR) luciferase assay, Frizzled-6 (FZD6) was confirmed to be a direct target of miR-199a/b in GC cells. siRNA-mediated depletion of FZD6 enhanced the motility of SNU601 cells, and addback of FZD6 restored cancer cell motility stimulated by miR-199a/b. In conclusion, miR-199a/b promotes DGC progression by targeting FZD6, implying that miR-199a/b can be used as prognostic and diagnostic biomarkers for the disease.

Cancer-associated fibroblasts (CAFs) play a substantial role in promoting cancer cell motility, drug resistance, angiogenesis, and metastasis; therefore, extensive research has been conducted to determine their mode of activation. We aimed to identify whether miRNA-200 (miR-200), a widely recognized suppressor of epithelial-mesenchymal transition, prevents CAFs from promoting cancer progression. Overexpression of miR-200 prevented CAFs from promoting lung cancer cell migration, invasion, tumorigenicity, and metastasis. Additionally, miR-200 suppressed the ability of CAFs to recruit and polarize macrophages toward the M2 phenotype, as well as the migration and tube formation of vascular endothelial cells. NRP2, a co-receptor of vascular endothelial growth factor receptor (VEGFR), was confirmed to be a target of miR-200, which mediates the functional activity of miR-200 in CAFs. NRP2-VEGFR signaling facilitates the secretion of VEGF-D and pleiotrophin from CAFs, leading to the activation of cancer cell migration and invasion. These findings suggest that miR-200 remodels CAFs to impede cancer progression and metastasis and that miR-200 and NRP2 are potential therapeutic targets in the treatment of lung cancer.

Abstract Background This study aimed to investigate how aging alters the homeostasis of the colonic intestinal epithelium and regeneration after tissue injury using organoid models and to identify its underlying molecular mechanism. Methods To investigate aging-related changes in the colonic intestinal epithelium, we conducted organoid cultures from old (older than 80 weeks) and young (6–10 weeks) mice and compared the number and size of organoids at day 5 of passage 0 and the growth rate of organoids between the two groups. Results The number and size of organoids from old mice was significantly lower than that from young mice (p

Abstract Tumor cells can respond to therapeutic agents by morphologic alternations including formation of tunneling nanotubes. Using tomographic microscope, which can detect the internal structure of cells, we found that mitochondria within breast tumor cells migrate to an adjacent tumor cell through a tunneling nanotube. To investigate the relationship between mitochondria and tunneling nanotubes, mitochondria were passed through a microfluidic device that mimick tunneling nanotubes. Mitochondria, through the microfluidic device, released endonuclease G (Endo G) into adjacent tumor cells, which we referred to herein as unsealed mitochondria. Although unsealed mitochondria did not induce cell death by themselves, they induced apoptosis of tumor cells in response to caspase‐3. Importantly, Endo G‐depleted mitochondria were ineffective as lethal agents. Moreover, unsealed mitochondria had synergistic apoptotic effects with doxorubicin in further increasing tumor cell death. Thus, we show that the mitochondria of microfluidics can provide novel strategies toward tumor cell death.

Abstract Background Since colon cancer stem cells (CSCs) play an important role in chemoresistance and in tumor recurrence and metastasis, targeting of CSCs has emerged as a sophisticated strategy for cancer therapy. α-mangostin (αM) has been confirmed to have antiproliferative and apoptotic effects on cancer cells. This study aimed to evaluate the selective inhibition of αM on CSCs in colorectal cancer (CRC) and the suppressive effect on 5-fluorouracil (5-FU)-induced CSCs. Methods The cell viability assay was performed to determine the optimal concentration of αM. A sphere forming assay and flow cytometry with CSC markers were carried out to evaluate the αM-mediated inhibition of CSCs. Western blot analysis and quantitative real-time PCR were performed to investigate the effects of αM on the Notch signaling pathway and colon CSCs. The in vivo anticancer efficacy of αM in combination with 5-FU was investigated using a xenograft mouse model. Results αM inhibited the cell viability and reduced the number of spheres in HT29 and SW620 cells. αM treatment decreased CSCs and suppressed the 5-FU-induced an increase in CSCs on flow cytometry. αM markedly suppressed Notch1, NICD1, and Hes1 in the Notch signaling pathway in a time- and dose-dependent manner. Moreover, αM attenuated CSC markers CD44 and CD133, in a manner similar to that upon DAPT treatment, in HT29 cells. In xenograft mice, the tumor and CSC makers were suppressed in the αM group and in the αM group with 5-FU treatment. Conclusion This study shows that low-dose αM inhibits CSCs in CRC and suppresses 5-FU–induced augmentation of CSCs via the Notch signaling pathway.

Vimentin is a type III intermediate filament protein expressed in cells of mesenchymal origin. Vimentin has been thought to function mainly as a structural protein and roles of vimentin in other cellular processes have not been extensively studied. Our current study aims to reveal functions of vimentin in macrophage foam cell formation, the critical stage of atherosclerosis. We demonstrated that vimentin null (Vim–/–) mouse peritoneal macrophages take up less oxidized LDL (oxLDL) than vimentin wild type (Vim+/+) macrophages. Despite less uptake of oxLDL in Vim–/– macrophages, Vim+/+ and Vim–/– macrophages did not show difference in expression of CD36 known to mediate oxLDL uptake. However, CD36 localized in plasma membrane was 50% less in Vim–/– macrophages than in Vim+/+ macrophages. OxLDL/CD36 interaction induced protein kinase A (PKA)-mediated vimentin (Ser72) phosphorylation. Cd36–/– macrophages did not exhibit vimentin phosphorylation (Ser72) in response to oxLDL. Experiments using phospho-mimetic mutation of vimentin revealed that macrophages with aspartate-substituted vimentin (V72D) showed more oxLDL uptake and membrane CD36. LDL receptor null (Ldlr–/–) mice reconstituted with Vim–/– bone marrow fed a western diet for 15 weeks showed 43% less atherosclerotic lesion formation than Ldlr–/– mice with Vim+/+ bone marrow. In addition, Apoe–/–Vim–/– (double null) mice fed a western diet for 15 weeks also showed 57% less atherosclerotic lesion formation than Apoe–/– and Vim+/+mice. We concluded that oxLDL via CD36 induces PKA-mediated phosphorylation of vimentin (Ser72) and phosphorylated vimentin (Ser72) directs CD36 trafficking to plasma membrane in macrophages. This study reveals a function of vimentin in CD36 trafficking and macrophage foam cell formation and may guide to establish a new strategy for the treatment of atherosclerosis.

MicroRNA transcriptomes from fresh tumors and the adjacent normal tissues were profiled in 10 Korean patients diagnosed with lung adenocarcinoma using a next-generation sequencing (NGS) technique called miRNA-seq. The sequencing quality was assessed using FastQC, and low-quality or adapter-contaminated portions of the reads were removed using Trim Galore. Quality-assured reads were analyzed using miRDeep2 and Bowtie. The abundance of known miRNAs was estimated using the reads per million (RPM) normalization method. Subsequently, using DESeq2 and Wx, we identified differentially expressed miRNAs and potential miRNA biomarkers for lung adenocarcinoma tissues compared to adjacent normal tissues, respectively. We defined reliable miRNA biomarkers for lung adenocarcinoma as those detected by both methods. The miRNA-seq data are available in the Gene Expression Omnibus (GEO) database under accession number GSE196633, and all processed data can be accessed via the Mendeley data website.

A fibrotic stroma accumulates in advanced cancers, and invasive cancer cells migrate along collagen fibers that facilitate dissemination from the primary tumor. However, the ways in which tumor cells govern these processes remain unclear. Here, we report that the epithelial-mesenchymal transition–activating transcription factor ZEB1 increased type I collagen (Col1) secretion and enhanced tumor cell adherence to Col1. Mechanistically, ZEB1 increased the levels of α1β1 integrin (encoded by Itga1 and Itgb1) by inhibiting PP2A activity, which reduced nuclear accumulation of HDAC4 and, thereby, derepressed Itga1 gene transcription. In parallel, ZEB1 relieved the miRNA-148a-mediated silencing of Itga1. High levels of Itga1 enhanced tumor cell adherence to Col1 and were essential for Col1-induced tumor growth and metastasis. Furthermore, ZEB1 enhanced Col1 secretion by increasing the expression of a kinesin protein that facilitated transport and secretion of Col1-containing vesicles. Our findings elucidate a transcriptional mechanism by which lung adenocarcinoma cells coordinate a collagen deposition and adhesion process that facilitates tumor progression.

The HfO2 nanoparticles and the nanocomposites of HfO2-graphene (10, 30, and 50 wt%) were prepared via precipitation and simple mixing method. The XRD pattern confirmed the presence of monoclinic HfO2 and hexagonal graphene in the nanocomposite. Raman spectroscopy studies revealed the formation of HfO2-graphene nanocomposite. According to SEM and TEM images the HfO2, NPs are spherical, and their size is less than 10 nm, anchored on the surface of the graphene sheets. The EDX spectrum shows carbon, oxygen, and HfO2 and reveals the formation of the HfO2-graphene nanocomposite. The UV-vis absorption spectra show the optical properties of synthesized HfO2-graphene nanocomposite. The study examines the influence of different ratios of the addition of graphene on the photocatalytic activity of HfO2-graphene. It was found that the HfO2-graphene (50 wt%) 40 mg nanocomposite exhibits enhanced photocatalytic activity than the bare HfO2 towards the methylene blue photodegradation, an aromatic pollutant in water under UV light irradiation, which can be applied optimally for individually wastewater management system. The HfO2-graphene (50 wt%) photocatalyst degrades 81 ± 2% of tetracycline in 180 min, implying that tetracycline can be degraded more efficiently under UV light. The enhancement in photocatalytic activity under UV light illumination can be attributed to the effective separation of photogenerated electrons, inhibiting recombination in the HfO2-graphene composite.

Summary: Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.

Cancer: Regulatory micro-RNAs could combine for treatment Exploring the effects of three similar small RNA molecules called micro-RNAs (miRNAs) that can restrict the activity of specific genes reveals how they might be used in cancer treatment. RNA is best known as messenger RNA, which carries a copy of a gene’s information into the cell cytoplasm to direct protein manufacture. Many small RNAs play less well-known but crucial roles by binding to messenger RNA molecules to regulate their activity. Researchers in South Korea and USA, led by Young-Ho Ahn at Ewha Womans University in Seoul, investigated how these miRNAs can suppress lung cancer in mice. Their results reveal details of how the miRNAs inhibit the expression of specific tumor-supporting genes. They suggest that three of the RNAs administered together might treat cancer more effectively than using only one as in previous trials.

Circular RNAs (circRNAs) are single-stranded RNAs with a covalently closed-loop structure that increases their stability; thus, they are more advantageous to use as liquid biopsy markers than linear RNAs. circRNAs are thought to be generated by back-splicing of pre-mRNA transcripts, which can be facilitated by reverse complementary sequences in the flanking introns and trans-acting factors, such as splicing regulatory factors and RNA-binding factors. circRNAs function as miRNA sponges, interact with target proteins, regulate the stability and translatability of other mRNAs, regulate gene expression, and produce microproteins. circRNAs are also found in the body fluids of cancer patients, including plasma, saliva, urine, and cerebrospinal fluid, and these “circulating circRNAs” can be used as cancer biomarkers. In lung cancer, some circulating circRNAs have been reported to regulate cancer progression and drug resistance. Circulating circRNAs have significant diagnostic value and are associated with the prognosis of lung cancer patients. Owing to their functional versatility, heightened stability, and practical applicability, circulating circRNAs represent promising biomarkers for lung cancer diagnosis, prognosis, and treatment monitoring.

Abstract Urothelial bladder carcinoma (UBC) is characterized by a large number of genetic alterations. DNA from urine is a promising source for liquid biopsy in urological malignancies. We aimed to assess the availability of cell-free DNA (cfDNA) and exosomal DNA (exoDNA) in urine as a source for liquid biopsy in UBC. We included 9 patients who underwent surgery for UBC and performed genomic profiling of tumor samples and matched urinary cfDNA and exoDNA. For mutation analysis, deep sequencing was performed for 9 gene targets and shallow whole genome sequencing (sWGS) was used for the detection of copy number variation (CNV). We analyzed whether genetic alteration in tumor samples was reflected in urinary cfDNA or exoDNA. To measure the similarity between copy number profiles of tumor tissue and urinary DNA, the Pearson’s correlation coefficient was calculated. We found 17 somatic mutations in 6 patients. Of the 17 somatic mutations, 14 and 12 were identified by analysis of cfDNA and exoDNA with AFs of 56.2% and 65.6%, respectively. In CNV analysis using sWGS, although the mean depth was 0.6X, we found amplification of MDM2, ERBB2, CCND1 and CCNE1, and deletion of CDKN2A, PTEN and RB1, all known to be frequently altered in UBC. CNV plots of cfDNA and exoDNA showed a similar pattern with those from the tumor samples. Pearson’s correlation coefficients of tumor vs. cfDNA (0.481) and tumor vs. exoDNA (0.412) were higher than that of tumor vs. normal (0.086). We successfully identified somatic mutations and CNV in UBC using urinary cfDNA and exoDNA. Urinary exoDNA could be another source for liquid biopsy. Also, CNV analysis using sWGS is an alternative strategy for liquid biopsy, providing data from the whole genome at a low cost.

Signal transducer and activator of transcription 6 (STAT6) promotes an anti-inflammatory process by inducing the development of M2 macrophages. We investigated whether modulating STAT6 activity in macrophages using AS1517499, the specific STAT6 inhibitor, affects the restoration of homeostasis after an inflammatory insult by regulating PPARγ expression and activity. Administration of AS1517499 suppressed the enhanced STAT6 phosphorylation and nuclear translocation observed in peritoneal macrophages after zymosan injection. In addition, AS1517499 delayed resolution of acute inflammation as evidenced by enhanced secretion of pro-inflammatory cytokines, reduced secretion of anti-inflammatory cytokines in PLF and supernatants from peritoneal macrophages, and exaggerated neutrophil numbers and total protein levels in PLF. We demonstrate temporal increases in annexin A1 (AnxA1) protein and mRNA levels in peritoneal lavage fluid (PLF), peritoneal macrophages, and spleen in a murine model of zymosan-induced acute peritonitis. In vitro priming of mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages with AnxA1 induced STAT6 activation with enhanced PPARγ expression and activity. Using AS1517499, we demonstrate that inhibition of STAT6 activation delayed recovery of PPARγ expression and activity, as well as impaired efferocytosis. Taken together, these results suggest that activation of the STAT6 signaling pathway mediates PPARγ expression and activation in macrophages to resolve acute inflammation.

Abstract Cancer-associated fibroblasts (CAFs) regulate diverse intratumoral biological programs and can promote or inhibit tumorigenesis, but those CAF populations that negatively impact the clinical outcome of lung cancer patients have not been fully elucidated. Because Thy-1 (CD90) marks CAFs that promote tumor cell invasion in a murine model of KrasG12D–driven lung adenocarcinoma (KrasLA1), here we postulated that human lung adenocarcinomas containing Thy-1+ CAFs have a worse prognosis. We first examined the location of Thy-1+ CAFs within human lung adenocarcinomas. Cells that co-express Thy-1 and α-smooth muscle actin (αSMA), a CAF marker, were located on the tumor periphery surrounding collectively invading tumor cells and in perivascular regions. To interrogate a human lung cancer database for the presence of Thy-1+ CAFs, we isolated Thy-1+ CAFs and normal lung fibroblasts (LFs) from the lungs of KrasLA1 mice and wild-type littermates, respectively, and performed global proteomic analysis on the murine CAFs and LFs, which identified 425 proteins that were differentially expressed. Used as a probe to identify Thy-1+ CAF-enriched tumors in a compendium of 1,586 lung adenocarcinomas, the presence of the 425-gene signature predicted a significantly shorter survival. Thus, Thy-1 marks a CAF population that adversely impacts clinical outcome in human lung cancer.

Artificial neural network-based analysis has recently been used to predict clinical outcomes in patients with solid cancers, including lung cancer. However, the majority of algorithms were not originally developed to identify genes associated with patients’ prognoses. To address this issue, we developed a novel prognosis-related feature selection framework called Cascaded Wx (CWx). The CWx framework ranks features according to the survival of a given cohort by training neural networks with three different high- and low-risk groups in a cascaded fashion. We showed that this approach accurately identified features that best identify the patients’ prognoses, compared to other feature selection algorithms, including the Cox proportional hazards and Coxnet models, when applied to The Cancer Genome Atlas lung adenocarcinoma (LUAD) transcriptome data. The prognostic potential of the top 100 genes identified by CWx outperformed or was comparable to those identified by the other methods as assessed by the concordance index (c-index). In addition, the top 100 genes identified by CWx were found to be associated with the Wnt signaling pathway, providing biologically relevant evidence for the value of these genes in predicting the prognosis of patients with LUAD. Further analyses of other cancer types showed that the genes identified by CWx had the highest prognostic values according to the c-index. Collectively, the CWx framework will potentially be of great use to prognosis-related biomarker discoveries in a variety of diseases.

The signal transducer and activator of transcription 6 (STAT6) transcription factor promotes activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway in macrophages. Little is known about the effect of proximal signal transduction leading to PPARγ activation for the resolution of acute inflammation. Here, we studied the role of STAT6 signaling in PPARγ activation and the resolution of acute sterile inflammation in a murine model of zymosan-induced peritonitis. First, we showed that STAT6 is aberrantly activated in peritoneal macrophages after zymosan injection. Utilizing STAT6−/− and wild-type (WT) mice, we found that STAT6 deficiency further enhanced zymosan-induced proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and macrophage inflammatory protein-2 in peritoneal lavage fluid (PLF) and serum, neutrophil numbers and total protein amount in PLF, but reduced proresolving molecules, such as IL-10 and hepatocyte growth factor, in PLF. The peritoneal macrophages and spleens of STAT6−/− mice exhibited lower mRNA and protein levels of PPARγ and its target molecules over the course of inflammation than those of WT mice. The deficiency of STAT6 was shown to impair efferocytosis by peritoneal macrophages. Taken together, these results suggest that enhanced STAT6 signaling results in PPARγ-mediated macrophage programming, contributing to increased efferocytosis and inflammation resolution.