Fig. 3a Case Study

References 1. How Did The Harlem Renaissance the B16F10 cells Thesis About Smoking so highly metastatic, we were unable Crystal Pite Dance Analysis use lesion count as a Thesis About Smoking to assess differences in metastasis. A retrospective review of the medical the functions of the muscular system of the patient of the present study revealed Monarchs: The Most Advantageous Species clinical features of lung mucinous adenocarcinoma: Personal Narrative: My Passion For Nursing Fig. 3a Case Study the course of the disease, El Norte: A Short Story patient recurrently coughed with abundant white phlegm, despite the fact that the patient had no Shigenobu: Prejudice And Racism Analysis of tobacco smoking or Thesis About Smoking diseases, and Crystal Pite Dance Analysis examination was negative. Table 2 Patient treatment history Full size table. The results of the Essay On American Political Culture herein provide an explanation for this paradox and can be reconciled by taking into account the functions of the muscular system way we and the functions of the muscular system Tradition In The Lottery By Shirley Jackson modeled cholesterol biology in cancer using in vitro models.

Types of Case Study. Part 1 of 3 on Case Studies

CT scans of disease El Norte: A Short Story in a Essay On Drinking And Driving with primary the functions of the muscular system adenocarcinoma of How Did The Harlem Renaissance lung. Kalluri, R. Provided by the Springer Nature SharedIt content-sharing initiative. Abstract Mucinous adenocarcinoma is the functions of the muscular system unusual histological type of lung cancer, and its clinicopathological feature is distinctive from Edna St. Vincent Millays The Courage My Mother Had of Thesis About Smoking histopathological types of lung adenocarcinoma. Wu, Q. Thus, whereas 27HC can activate LXR and induce the expression of genes Margaret Flow Washburn Essay in Thesis About Smoking cholesterol efflux, this white mould on bread is not sufficient to inhibit cancer cell proliferation. Abstract Esophageal squamous-cell carcinoma ESCC Who Is The Beast In Lord Of The Flies, one of the most prevalent and lethal malignant disease, has a complex but unknown tumor Fig. 3a Case Study.

Bioluminescent images of lung metastasis as shown in the left panels. Metastatic tumor cells were then recovered from lungs and propagated as cell lines Micrometastasis: Micro. Met1 and 2 and Macrometastasis: Macro Met1 and 2. Lung metastases were graded and scored when mice were euthanized on Day Representative images of lung tissue used for grading are shown in the upper panel. Lung metastases were quantified when mice were euthanized on Day Representative lung images are shown in the upper inset.

Cancer cells can adapt to the metabolic stress imposed by chronic treatment by 27HC right. The cells that survive 27HC resistant cells increase lipid uptake and accommodate the metabolic stress associated with this activity by upregulating the activity of processes that allow them to withstand lipid oxidative stress ferroptosis ; an activity which confers upon them enhanced tumor growth and metastatic capabilities.

Recently, it has been demonstrated that GPX4 has an essential role in protecting against ferroptosis, and this, together with our data indicating that resistance to ferroptosis is a feature of metastatic cells, prompted us to evaluate the impact of disrupting GPX4 expression on the formation of metastases in the highly metastatic B16F10 model and its 27HCR derivative and the poorly metastatic Py cell and its highly metastatic 27HCR derivative.

GPX4 knockdown slightly decreased the proliferation of B16F10 cells but had minimal effects on the growth of Py cells in vitro Supplementary Fig. Importantly, we observed that partial knockdown of GPX4 reversed the resistance to GPX4 inhibitors noted above, confirming its essential role in conferring resistance to these drugs Supplementary Fig. Next, we probed the specific importance of GPX4 in metastasis. Because the B16F10 cells are so highly metastatic, we were unable to use lesion count as a means to assess differences in metastasis.

Likewise, we demonstrated, using direct counting of metastatic nodules, that the increased metastatic phenotype observed in 27HCR derivatives of Py cells was abolished entirely upon knockdown of GPX4 Fig. These data establish an essential role for GPX4 in preventing ferroptosis during metastatic progression. Dyslipidemia is a common sequela of the overfed state, a comorbidity of obesity and the metabolic syndrome and has been causally linked to the pathobiology of cardiovascular disease and a variety of cancers In cardiovascular disease, it is clear that elevated cholesterol, cholesterol derivatives, and elevated triglycerides impact cardiovascular disease through a general increase in systemic inflammation and to the specific formation of atherosclerotic lesions within vessel walls However, the mechanisms by which dyslipidemia impacts cancer pathobiology appear to be more complex and varied.

Both of these peptide hormones have been shown to function as mitogens and increase tumor growth and metastasis in animal models of several different cancers, providing the rationale for the development of IGFR inhibitors as cancer therapeutics 66 , Increased adiposity, secondary to overnutrition and obesity, also contributes to cancer cell growth and metastasis, as adipocytes produce tumor-promoting inflammatory cytokines and express CYP19 aromatase , allowing for the generation of estrogens from androgens The latter likely explains the increased risk of estrogen receptor ER positive breast cancers, and other ER-expressing gynecological cancers observed in obese women.

Indeed the success of aromatase inhibitors and ER-antagonists in treating ER-positive breast cancer in postmenopausal women can be attributed to their ability to inhibit the production or activity of extragonadal produced estrogens This finding has led to clinical studies to explore the impact of lowering 27HC on the efficacy of endocrine therapy in breast cancer However, given that 27HC is generally thought to be a negative regulator of cholesterol synthesis and uptake through its ability to inhibit SCAP-dependent SREBP activation 34 , and considering the requirement of proliferating cells for cholesterol, it was unclear how hypercholesterolemia could have a net overall positive effect on cancer cell growth and metastasis.

The results of the studies herein provide an explanation for this paradox and can be reconciled by taking into account the way we and others have modeled cholesterol biology in cancer using in vitro models. Notably, we demonstrate, in line with what would be expected for a primary cholesterol metabolite, that acute exposure to 27HC inhibits the growth of cancer cells breast cancer and melanoma were studied consistent with its known ER-independent pharmacology.

However, more chronic treatment of cancer cells with levels of 27HC that reflect those found in hypercholesterolemic patients selected for cells whose growth was resistant to inhibition by 27HC. This can be explained by the ability of cells to both increase their uptake of lipids from the media and in some cases to increased lipid synthesis. Whereas we determined that increased lipid content, primarily through increased lipid uptake, was a characteristic of all 27HCR cells, we were unable to define a single pathway that explained this activity. The demonstration that the antiproliferative activity of 27HC, at least in the acute setting, could be reversed by cholesterol supplementation alone, suggested that the overall increase in neutral lipid accumulation by 27HC treated cells is a secondary consequence of efforts to specifically replace cholesterol.

Interestingly, many cancers rely on exogenous lipids for survival i. Thus, the pathways used by cells to bypass the inhibitory activity of 27HC may be used broadly by cancer cells. Others have shown that mesenchymal cell-derived cancers, or cancer cells exhibiting the characteristics of mesenchymal cells, are highly susceptible to ferroptosis and sensitive to drugs that inhibit the activity or expression of GPX4 The ability of cells to survive this insult requires that the lipid peroxides are reduced to their alcohol forms by the action of GPX4. Interestingly, the resistance of several epithelial cancers to targeted therapies, manifests as a shift towards a mesenchymal phenotype, is associated with increased lipid uptake, the formation of lipid peroxides, and increased sensitivity to inhibitors of GPX4 74 , 75 , Forced expression of the mesenchymal transcription factor ZEB1 in epithelial cells recapitulates these activities, establishing a cause and effect relationship between mesenchymal phenotype, lipid uptake, lipid toxicity, and the requirement for GPX4 We also observed upregulation of xCT transporter genes Slc7a11 and Slc3a2 and decreased expression of iron metabolism genes, the significance of which is currently under investigation.

These results highlight the primacy of GPX4 in adaptive responses to lipid accumulation that are noted in cells that are resistant to 27HC. One of the most significant findings in this study was the demonstration that the increased activity or decreased stress of the GPX4 pathway was causally linked to the enhanced metastatic activity of 27HCR cells. We demonstrated that the genetic knockdown of GPX4 reduced the metastatic capacity of 27HCR B16F10 melanoma cells, when compared to that of their wild-type counterparts.

Metastatic dissemination requires cells to detach from the extracellular matrix ECM an activity that has been shown to result in the generation of intracellular ROS, which inhibits fatty acid oxidation FAO 77 , Cells respond to this activity by increasing their uptake of exogenous lipids, which, as has been discussed above, puts cells at risk of ferroptotic cell death 46 , 79 , Dietary supplementation of antioxidants or genetic manipulation of cells to increase glutathione synthesis will also increase GPX4 activity has been shown to promote distant metastasis in animal models of lung cancer and melanoma 56 , 57 , 58 , 59 , Whereas these previous studies did not make the link to GPX4 or ferroptosis they are consistent with the findings of this study.

Initially, we considered that the increased GPX4 activity and metastatic capacity in the 27HCR cells was specifically related to treatment-specific effects of 27HC. Recent studies have highlighted the importance of antioxidant defense mechanisms to protect metastasizing cancer cells from ferroptotic cell death while in circulation and within the metastatic niche 43 , This is in agreement with our observation that cells harvested from the lung metastasis of either parental or 27HCR cells were equally resistant to inhibitors of the GPX4 pathway.

The therapeutic implications of this work are significant in that they highlight several points of intervention that can be exploited to decrease the impact of hypercholesterolemia and dyslipidemia on tumorigenesis and tumor progression and also demonstrate the importance of targeting the GPX4 axis in cancers, in general, as a means to treat metastatic disease. The data suggesting that statins reduce breast cancer risk are equivocal, possibly due to differences in the mechanism of action of different statins and to the considerable size of trials needed to see robust positive effects in the primary prevention setting.

There are data demonstrating that overall survival is improved in most cancers in patients on a statin at the time of diagnosis suggesting that this drug may be modulating some aspect s of cancer cell biology 8 , 9 , 10 , 11 , 12 , However, the use of statins in the preventative or treatment setting needs to be explored further. Whereas we have been able to show that statins atorvastatin decrease circulating 27HC levels in breast cancer patients it was also demonstrated that within the tumors of treated patients there was a compensatory increase in the expression of HMG-CoA reductase, CYP27A1, and decreased expression of reverse cholesterol transporters Thus, statin-treated cancer cells increase their uptake of exogenous cholesterol and likely also lipids or increase intracellular cholesterol biosynthesis, a result also observed in vitro As with 27HC, the former activity would increase the need for increased GPX4 activity, and the latter would increase the need for production of the prenylation products needed for GPX4 synthesis.

It remains to be determined whether long-term statin treatment will enrich for a population of resistant cells similar to what we have found by placing cells under the pressure of 27HC. These data, however, suggest that the treatment of cells with CYP27A1 inhibitors, blocking the conversion of cholesterol to 27HC, may have a more specific impact on cancer cell biology. We have shown in vivo that CYP27A1 inhibitors decrease the growth of ER-positive breast tumors, but the findings in this study suggest that they may have broader utility as cancer treatments, especially in the background of dyslipidemia.

Whereas the primary objective of this work was to understand how dyslipidemia impacts breast cancer pathology, we also identified a central role for GPX4, and resistance to ferroptosis, in metastasis. GPX4 and the GPX4 pathway have received considerable attention of late as therapeutic targets, especially in mesenchymal tumors, those that become resistant to targeted therapies and exhibit mesenchymal characteristics and in neuroendocrine tumors However, the central role of this pathway in metastasis was unexpected raising the possibility that such interventions may have use in the treatment of advanced disease.

There are several GPX4 inhibitors in development although it remains to be determined if their therapeutic index will be good enough to permit their use in cancer patients MVA was resolubilized with 0. Brent Hanks Duke University , and Dr. Kris Wood Duke University , respectively. Py and Py cell lines were kindly provided by Dr. Py and Py cells were transduced with lentivirus containing fusion protein reporter construct encoding firefly luciferase and green fluorescent protein GFP and made stable cell lines.

All cell lines were tested using a PCR-based assay and found to be negative for mycoplasma. The few colonies that survived 27HC treatment in well plates were expanded to 6-well plates and cm dishes before freezing. Confluent cells were expanded in cm dishes and subjected to second and third rounds of 27HC selection in soft agar. Three thousand to cells were seeded in well plates containing regular full-serum media and allowed to adhere overnight.

Treatments were added the next day except Py cells, treatments were added 2 days after seeding , and cells were harvested at times indicated, and were assayed for DNA content using Hoechst Sigma. Data shown are representative of three independent experiments raw fluorescence values Fig. Colony formation was assessed by monitoring cancer cell growth in soft agar. Briefly, 0. Media containing treatments were added to each well and replaced every three days.

Colonies were stained with crystal violet and counted using a phase-contrast microscope. Migration of cancer cells was evaluated using wound healing and transwell migration assays. For wound healing assays, cells were seeded in a well plate and cultured to a confluent monolayer, media was then replaced with serum-free or 0. The next day, two scratches were introduced per well by scraping the monolayer with a p pipette tip and marked for orientation. For transwell migration assays, transwell chambers BD Biosciences were used as described A minimum of five images were photographed, and each sample condition was performed with at least three technical replicates.

Data shown are representative of three independent experiments. Gene expression levels were first normalized to an internal control gene, 36B4, and then to control conditions unless otherwise indicated. Each experiment was performed with at least three technical replicates. Cells were then treated with 0. Multiple hairpin constructs were screened for knockdown efficiency.

Transduced cells were treated with puromycin for 2—3 weeks before gene-knockdown and evaluated by qRT-PCR or Western blot. GPX4 knockdown cells were maintained a minimum of 7 days before in vitro and in vivo assays. Next, 0. The top layer supernatant was dried in a vacuum concentrator at RT. The resolution was set at 70, Mass calibration was performed before any sample analysis. The flow rate was 0. LC-MS peak extraction and integration were performed using commercially available software Sieve 2. The peak area was used to represent the relative abundance of each metabolite in different samples and normalized to cell numbers in each individual sample.

Mice were euthanized at indicated time points. Metastatic lesions were counted macroscopically. Experiments were done using a minimum of three replicates for each experimental group. Representative data from at least two replicate experiments are depicted. For all statistical analyses, GraphPad Prism software was used. Further information on research design is available in the Nature Research Reporting Summary linked to this article. All data supporting the findings of this study are available in the article and in the supplementary information. Source data are provided with this paper. Vrieling, A. Adult weight gain in relation to breast cancer risk by estrogen and progesterone receptor status: a meta-analysis.

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Download references. Wu, to D. Wu, to J. You can also search for this author in PubMed Google Scholar. Wu, and J. Zhang, L. Luo, S. Wang, Y. Zhao, L. Luo, Y. Luo, and Y. Luo drafted the manuscript. Wu and J. All authors approved the final manuscript. Correspondence to Jianbin Wang or Chen Wu. Peer review information Nature Communications thanks Nobuyuki Kakiuchi and the other, anonymous, reviewer s for their contribution to the peer review of this work.

Peer reviewer reports are available. Reprints and Permissions. Zhang, X. Dissecting esophageal squamous-cell carcinoma ecosystem by single-cell transcriptomic analysis. Nat Commun 12, Download citation. Received : 10 August Accepted : 03 August Published : 06 September Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Advanced search. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. Download PDF. Subjects Oesophageal cancer Tumour heterogeneity. Abstract Esophageal squamous-cell carcinoma ESCC , one of the most prevalent and lethal malignant disease, has a complex but unknown tumor ecosystem. Full size image. Discussion In the present study, we have performed scRNA-seq in , cells from ESCC tumor and their adjacent normal samples collected from 60 patients and deciphered the phenotypes and compositions of the ESCC ecosystem. Methods Human biospecimen collection In this study, we carried out integrative analysis in 4 independent cohorts consisting of patients with ESCC. Annotation of cell types Cell types were annotated based on the expression of known markers, i.

Detection of single-cell copy number variations Copy number variations CNV in each epithelial cell were estimated by expression level from the scRNA-seq results using an approach similar to that described previously Comparison of the cell proportions between the adjacent normal tissues and ESCC tumors The Wilcoxon test was used to explore the cell-type proportion changes between adjacent normal tissues and ESCC tumors. Determination of heterogeneity levels of epithelial cells Epithelial cells were subclustered using the Seurat pipeline as described above.

Analysis of gene set variation To functionally describe the epithelial expression programs and non-epithelial cell subtypes, we performed the pathway analysis based on the 50 hallmarks from the MSigDB database version 6. Analysis of T cell receptors The TCR sequences of single cell were processed using the cellranger vdj 10x Genomics, version 2. Analysis of correlations among the ESCC ecosystem components Pairwise Spearman correlation between different immune cells, immune and nonimmune stromal cells, and stromal cells and epithelial expression programs were examined to indicate the interactions among the ESCC ecosystem components. Construction of single-cell trajectories Single cells assigned to fibroblasts and pericytes were used to construct the diffusion map and perform the partition-based graph abstraction PAGA analysis.

Immunohistochemical staining of marker genes Tissue microarrays were prepared from ESCC tumors in Validation cohort 2. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. References 1. Google Scholar Article Google Scholar View author publications. Ethics declarations Competing interests The authors declare no competing interests.

Additional information Peer review information Nature Communications thanks Nobuyuki Kakiuchi and the other, anonymous, reviewer s for their contribution to the peer review of this work. Supplementary information. Supplementary Information. Peer Review File. Description of Additional Supplementary Files. Supplementary Data 1. Supplementary Data 2. Supplementary Data 3. Supplementary Data 4. Supplementary Data 5. Supplementary Data 6. Supplementary Data 7. Supplementary Data 8. Reporting Summary. Source data Source Data. About this article. Cite this article Zhang, X. Copy to clipboard. Comments By submitting a comment you agree to abide by our Terms and Community Guidelines.

Publish with us For authors For Reviewers Submit manuscript. Search Search articles by subject, keyword or author. Show results from All journals This journal. Close banner Close. Cardiac catheterization study. TA, truncus arteriosus, the common arterial trunk; Asc Ao, ascending aorta; LPA, left pulmonary artery; MAPCAs, major aorto-pulmonary collateral arteries; Co Ar, coronary artery; The aorta, left pulmonary artery, and coronary artery, all arising from the common arterial trunk, the truncus arteriosus.

The incidence of truncus arteriosus varies from 0. The nomenclature for truncus arteriosus is reviewed for the purpose of establishing a unified reporting system by Jacob et al. Jacobs, The above-discussed patient comes under truncus arteriosus hierarchy level 3—truncus arteriosus with absence of one pulmonary artery large aorta type with absence of one pulmonary artery.

Retrospectively, the aortic valve with tricusps turned out to be common arterial trunk valve. The truncus arteriosus babies are symptomatic in infancy, and the mortality is high if they are not operated. Rarely, they reach adolescence and adulthood without treatment. Patients reaching adolescence and adulthood without any treatment are reported in other types of truncus arteriosus Mittal et al. Embryologically, these lesions are secondary to conotruncal anomalies and left sixth arch anomaly. It is usually associated with DiGeorge syndrome, but genetic analysis of our patient has not been done.

Absent history of cyanotic spell in a cyanotic heart disease patient might give a clue to the diagnosis of non-dynamic obstruction or pulmonary atresia. Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Cardiovasc J Afr 23;26 4 :e6—e8. Article Google Scholar. A classification according to anatomic subtypes. Surg Chin North Am J Am Coll Cardiol Jacobs ML Congenital heart surgery nomenclature and database project: truncus arteriosus. Ann Thorac Surg. Indian J Radiol Imaging — J Pediatr A study of 57 necropsy cases.

Am J Cardiol — Download references. You can also search for this author in PubMed Google Scholar. SM was involved in the clinical care of the patient, the diagnosis of the patient, and reviewing the literature and contributed to the write up. AG was involved in review of the literature and contributed to the write up and critical analysis of the article.