Tobacco treatment in the setting of lung cancer screening.

PURPOSE OF REVIEW: Lung cancer screening by low-dose CT is an increasingly implemented preventive medicine tool. Screening for lung cancer is incomplete without addressing problematic tobacco use, the greatest modifiable risk factor in the development of lung cancer. This review describes recent work related to lung cancer screening and treatment of tobacco use in that context. RECENT FINDINGS: Implementation of lung cancer screening demonstrates socioeconomic disparities in terms of adherence to screening as well as likelihood of successful tobacco dependence treatment. Active tobacco dependence is a common comorbidity for patients undergoing lung cancer screening. The optimal implementation of tobacco dependence treatment in the context of lung cancer screening is still an area of active investigation. SUMMARY: Treatment of tobacco dependence at time of lung cancer screening is a major opportunity for clinicians to intervene to reduce the major modifiable risk factor for lung cancer, tobacco use. Providing comprehensive tobacco dependence treatment is most effective using combination pharmacologic and behavioral interventions. Practices providing comprehensive treatment will benefit from accurate documentation for billing and coding and supplementing with external resources such as state Quit Lines.

A Comparative Analysis of Online Versus in-Person Opioid Overdose Awareness and Reversal Training for First-Year Medical Students.

INTRODUCTION: Physicians trained in opioid use disorder (OUD) harm reduction can mitigate opioid overdose deaths by prescribing naloxone and educating patients about its use. Unfortunately, many physicians possess OUD stigma. Training during medical school presents an opportunity to reduce OUD stigma and improve opioid overdose reversal knowledge. This study assessed the efficacy of Opioid Overdose Awareness and Reversal Training (OOART) and evaluated the equivalency of the online and in-person OOART. Methods: Voluntary training was delivered to first-year medical (M1) students at one medical school. In 2018 and 2019, 29 and 68 M1 students, respectively, received in-person OOART training and completed pre- and post-training surveys. In 2020, 62 students participated in online OOART training, of which 53 completed both pre- and post-training surveys. Results: All three opioid overdose Knowledge questions showed significant improvements between pre- and post-training survey responses. For Attitude questions, six of eleven questions in 2019 and 2020 and four of eleven questions in 2018 had statistically significant improvements between pre- and post-training survey responses. There were no statistical differences between in-person and online post-training survey results for two out of the three Knowledge questions and all 11 Attitude questions. Conclusions: This study demonstrates that our OOART was effective in increasing opioid overdose reversal knowledge and reducing OUD stigma. There was no meaningful difference in outcomes between the training modalities. These results support the future expansion of online and in-person OOART to a larger population of medical students.

Publisher Correction: 3D Shape Modeling for Cell Nuclear Morphological Analysis and Classification.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

3D Shape Modeling for Cell Nuclear Morphological Analysis and Classification.

Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with pathological conditions such as cancer. However, dimensionality of imaging data, together with a great variability of nuclear shapes, presents challenges for 3D morphological analysis. Thus, there is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analysis. We propose a new approach that combines modeling, analysis, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. We used robust surface reconstruction that allows accurate approximation of 3D object boundary. Then, we computed geometric morphological measures characterizing the form of cell nuclei and nucleoli. Using these features, we compared over 450 nuclei with about 1,000 nucleoli of epithelial and mesenchymal prostate cancer cells, as well as 1,000 nuclei with over 2,000 nucleoli from serum-starved and proliferating fibroblast cells. Classification of sets of 9 and 15 cells achieved accuracy of 95.4% and 98%, respectively, for prostate cancer cells, and 95% and 98% for fibroblast cells. To our knowledge, this is the first attempt to combine these methods for 3D nuclear shape modeling and morphometry into a highly parallel pipeline workflow for morphometric analysis of thousands of nuclei and nucleoli in 3D.

O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis.

Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked ß-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.

Characterization of Urothelial Cancer Circulating Tumor Cells with a Novel Selection-Free Method.

OBJECTIVE: To investigate circulating tumor cells (CTCs) as biomarkers of urothelial carcinoma (UC). To date, the majority of work on this topic has utilized the CellSearch test, which has limited sensitivity due to reliance on positive selection for the cell surface protein epithelial cell adhesion molecule (EpCAM). We used a novel selection-free method to enumerate and characterize CTCs across a range of UC stages. MATERIALS AND METHODS: Blood samples from 38 patients (9 controls, 8 nonmuscle invasive bladder cancer [NMIBC], 12 muscle-invasive bladder cancer [MIBC], and 9 metastatic UC) were processed with the AccuCyte-CyteFinder system. Slides were stained for the white blood cell markers CD45 and CD66b and the epithelial markers EpCAM and pancytokeratin. CTCs were defined as any cytokeratin postive and white blood cell marker negative cell. Separately, the more restrictive CellSearch definition was applied, with the additional requirement of EpCAM positivity. The Kruskal-Wallis ANOVA test compared CTC counts by stage. RESULTS: Greater than or equal to 1 CTC was detected in 2 of 8 (25%) patients with NMIBC, 7 of 12 (58%) with MIBC, and 6of 9 (67%) with metastatic disease. No control had CTCs. Comparing CTC counts between groups, the only statistically significant comparison was between controls and patients with metastatic UC (P = .009). With EpCAM positivity as a CTC requirement, no CTCs were detected in any patient with NMIBC, and only 2 (17%) patients with MIBC had CTCs. CTCs tended to be larger in metastatic patients. CONCLUSION: CTCs were detected at all UC stages and exhibited phenotypic diversity of cell size and EpCAM expression. EpCAM negative CTCs that would be missed with the CellSearch test were detected in patients with NMIBC and patients with MIBC.

The prostate metastasis suppressor gene NDRG1 differentially regulates cell motility and invasion.

Experimental and clinical evidence suggests that N-myc downregulated gene 1 (NDRG1) functions as a suppressor of prostate cancer metastasis. Elucidating pathways that drive survival and invasiveness of NDRG1-deficient prostate cancer cells can help in designing therapeutics to target metastatic prostate cancer cells. However, the molecular mechanisms that lead NDRG1-deficient prostate cancer cells to increased invasiveness remain largely unknown. In this study, we demonstrate that NDRG1-deficient prostate tumors have decreased integrin expression and reduced cell adhesion and motility. Our data indicate that loss of NDRG1 differentially affects Rho GTPases. Specifically, there is a downregulation of active RhoA and Rac1 GTPases with a concomitant upregulation of active Cdc42 in NDRG1-deficient cells. Live cell imaging using a fluorescent sensor that binds to polymerized actin revealed that NDRG1-deficient cells have restricted actin dynamics, thereby affecting cell migration. These cellular and molecular characteristics are in sharp contrast to what is expected after loss of a metastasis suppressor. We further demonstrate that NDRG1-deficient cells have increased resistance to anoikis and increased invasiveness which is independent of its elevated Cdc42 activity. Furthermore, NDRG1 regulates expression and glycosylation of EMMPRIN, a master regulator of matrix metalloproteases. NDRG1 deficiency leads to an increase in EMMPRIN expression with a concomitant increase in matrix metalloproteases and thus invadopodial activity. Using a three-dimensional invasion assay and an in vivo metastasis assay for human prostate xenografts, we demonstrate that NDRG1-deficient prostate cancer cells exhibit a collective invasion phenotype and are highly invasive. Thus, our findings provide novel insights suggesting that loss of NDRG1 leads to a decrease in actin-mediated cellular motility but an increase in cellular invasion, resulting in increased tumor dissemination which positively impacts metastatic outcome.

Nucleolin Staining May Aid in the Identification of Circulating Prostate Cancer Cells.

INTRODUCTION: Circulating tumor cells (CTCs) have great potential as circulating biomarkers for solid malignancies. Currently available assays for CTC detection rely on epithelial markers with somewhat limited sensitivity and specificity. We found that the staining pattern of nucleolin, a common nucleolar protein in proliferative cells, separates CTCs from white blood cells (WBCs) in men with metastatic prostate cancer. PATIENTS AND METHODS: Whole peripheral blood from 3 men with metastatic prostate cancer was processed with the AccuCyte CTC system (RareCyte, Seattle, WA). Slides were immunostained with 4',6-diamidino-2-phenylindole (DAPI), anti-pan-cytokeratin, anti-CD45/CD66b/CD11b/CD14/CD34, and anti-nucleolin antibodies and detected using the CyteFinder system. DAPI nucleolin colocalization and staining pattern wavelet entropy were measured with novel image analysis software. RESULTS: A total of 33,718 DAPI-positive cells were analyzed with the novel imaging software, of which 45 (0.13%) were known CTCs based on the established AccuCyte system criteria. Nucleolin staining pattern for segmentable CTCs demonstrated greater wavelet entropy than that of WBCs (median wavelet entropy, 6.86 × 107 and 3.03 × 106, respectively; P = 2.92 × 10-22; approximated z statistic = 9.63). Additionally, the total nucleolin staining of CTCs was greater than that of WBCs (median total pixel intensity, 1.20 × 105 and 2.55 × 104 integrated pixel units, respectively; P = 2.40 × 10-21; approximated z statistic = 9.41). CONCLUSION: Prostate cancer CTCs displayed unique nucleolin expression and localization compared to WBCs. This finding has the potential to serve as the basis for a sensitive and specific CTC detection method.

A surface tension magnetophoretic device for rare cell isolation and characterization.

The cancer community continues to search for an efficient and cost-effective technique to isolate and characterize circulating cells (CTCs) as a 'real-time liquid biopsy'. Existing methods to isolate and analyze CTCs require various transfer, wash, and staining steps that can be time consuming, expensive, and led to the loss of rare cells. To overcome the limitations of existing CTC isolation strategies, we have developed an inexpensive 'lab on a chip' device for the enrichment, staining, and analysis of rare cell populations. This device utilizes immunomagnetic positive selection of antibody-bound cells, isolation of cells through an immiscible interface, and filtration. The isolated cells can then be stained utilizing immunofluorescence or used for other downstream detection methods. We describe the construction and initial preclinical testing of the device. Initial tests suggest that the device may be well suited for the isolation of CTCs and could allow the monitoring of cancer progression and the response to therapy over time.

Circulating tumour cells as biomarkers of prostate, bladder, and kidney cancer.

Circulating tumour cells (CTCs) have been studied as biomarkers of a number of solid malignancies. Potential clinical applications for CTC analysis include early cancer detection, disease staging, monitoring for recurrence, prognostication, and to aid in the selection of therapy. In the field of urologic oncology, CTCs have been most widely studied as prognostic biomarkers of castration-resistant prostate cancer. Additionally, emerging data support a role for CTCs to help identify which patients are most likely to respond to novel androgen-pathway targeted therapies, such as abiraterone and enzalutamide. CTCs have also been studied as predictive biomarkers of bladder cancer, in particular as a means to identify patients whose disease has been clinically understaged. Less is known regarding CTCs in kidney cancer; this has been attributed to the fact that a minority of renal tumours express EpCAM, the epithelial cell surface protein commonly used by CTC assays for positive cell selection. However, alternative approaches using markers specific for kidney cancer are being explored.

A simple selection-free method for detecting disseminated tumor cells (DTCs) in murine bone marrow.

Bone metastasis is a lethal and incurable disease. It is the result of the dissemination of cancer cells to the bone marrow. Due to the difficulty in sampling and detection, few techniques exist to efficiently and consistently detect and quantify disseminated tumor cells (DTCs) in the bone marrow of cancer patients. Because mouse models represent a crucial tool with which to study cancer metastasis, we developed a novel method for the simple selection-free detection and quantification of bone marrow DTCs in mice. We have used this protocol to detect human and murine DTCs in xenograft, syngeneic, and genetically engineered mouse models. We are able to detect and quantify bone marrow DTCs in mice that do not have overt bone metastasis. This protocol is amenable not only for detection and quantification purposes but also to study the expression of markers of numerous biological processes or tissue-specificity.

Technical challenges in the isolation and analysis of circulating tumor cells.

Increasing evidence suggests that cancer cells display dynamic molecular changes in response to systemic therapy. Circulating tumor cells (CTCs) in the peripheral blood represent a readily available source of cancer cells with which to measure this dynamic process. To date, a large number of strategies to isolate and characterize CTCs have been described. These techniques, however, each have unique limitations in their ability to sensitively and specifically detect these rare cells. In this review we focus on the technical limitations and pitfalls of the most common CTC isolation and detection strategies. Additionally, we emphasize the difficulties in correctly classifying rare cells as CTCs using common biomarkers. As for assays developed in the future, the first step must be a uniform and clear definition of the criteria for assigning an object as a CTC based on disease-specific biomarkers.

A phased strategy to differentiate human CD14+monocytes into classically and alternatively activated macrophages and dendritic cells.

There are currently several in vitro strategies to differentiate human CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) into the M1 or M2 macrophage cell types. Each cell type is then verified using flow cytometric analysis of cell-surface markers. Human CD14(+) monocytes have the potential to differentiate into M1 and M2 macrophages, both of which demonstrate varying degrees of cell-surface antigen overlap. Using multiple surface markers with current macrophage polarization protocols, our data reveal several limitations of currently used methods, such as highly ambiguous cell types that possess cell-surface marker overlap and functional similarities. Utilizing interleukin-6 (IL-6) and two phases of cytokine exposure, we have developed a protocol to differentiate human monocytes into M1, M2, or dendritic cells (DCs) with greater efficiency and fidelity relative to macrophages and DCs that are produced by commonly used methods. This is achieved via alterations in cytokine composition, dosing, and incubation times, as well as improvements in verification methodology. Our method reliably reproduces human in vitro monocyte-derived DCs and macrophage models that will aid in better defining and understanding innate and adaptive immunity, as well as pathologic states.

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters.

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14(+) cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14(+) epithelial tumor cell clusters disseminate collectively to colonize distant organs.

Disseminated tumor cells and dormancy in prostate cancer metastasis.

It has been reported that disseminated tumor cells (DTCs) can be found in the majority of prostate cancer (PCa) patients, even at the time of primary treatment with no clinical evidence of metastatic disease. This suggests that these cells escaped the primary tumor early in the disease and exist in a dormant state in distant organs until they develop in some patients as overt metastases. Understanding the mechanisms by which cancer cells exit the primary tumor, survive the circulation, settle in a distant organ, and exist in a quiescent state is critical to understanding tumorigenesis, developing new prognostic assays, and designing new therapeutic modalities to prevent and treat clinical metastases.

Alternative CD44 splicing identifies epithelial prostate cancer cells from the mesenchymal counterparts.

An epithelial to mesenchymal transition (EMT) has been shown to be a necessary precursor to prostate cancer metastasis. Additionally, the differential expression and splicing of mRNAs has been identified as a key means to distinguish epithelial from mesenchymal cells by qPCR, western blotting and immunohistochemistry. However, few markers exist to differentiate between these cells by flow cytometry. We previously developed two cell lines, PC3-Epi (epithelial) and PC3-EMT (mesenchymal). RNAseq was used to determine the differential expression of membrane proteins on PC3-Epi/EMT. We used western blotting, qPCR and flow cytometry to validate the RNAseq results. CD44 was one of six membrane proteins found to be differentially spliced between epithelial and mesenchymal PC3 cells. Although total CD44 was positive in all PC3-Epi/EMT cells, PC3-Epi cells had a higher level of CD44v6 (CD44 variant exon 6). CD44v6 was able to differentiate epithelial from mesenchymal prostate cancer cells using either flow cytometry, western blotting or qPCR.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells.

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.

The presence of androgen receptor elements regulates ZEB1 expression in the absence of androgen receptor.

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that plays a central role in the epithelial to mesenchymal transition (EMT) of cancer cell lines. Studies on its regulation have mostly focused on the negative 3'UTR binding of miR200c. Interestingly, it has been previously reported that androgen receptor (AR) regulates ZEB1 expression in breast and prostate cancers. In order to validate this, various ZEB1 promoter deletions were cloned into a luciferase reporter system to elucidate the contribution of two putative androgen response elements (AREs). The in vivo contribution of AR was also assessed in cell lines after R1881 treatment using qPCR with prostate specific antigen (PSA) as the positive control. We discovered that AR upregulates the levels of expression of ZEB1 10-fold on a luciferase promoter that only contains the distal ARE. However, when the proximal ARE is included, no additional activation is apparent with AR or its hormone independent variant, AR-V7. Furthermore, we demonstrate here that a promoter construct containing both AREs activates transcription of ZEB1 even in the AR-null cell lines DU145 and PC3. Incubation of the AR-positive cell line, LNCaP with R1881, failed to substantially increase the expression levels of ZEB1. Despite the presence of AREs in the promoter region, it appears that ZEB1 expression can be induced even without AR. In addition, the region around the distal ARE is a potent repressor in AR-null cell lines.

Epithelial-mesenchymal transition in prostate cancer is associated with quantifiable changes in nuclear structure.

BACKGROUND: Prostate cancer progression is concomitant with quantifiable nuclear structure and texture changes as compared to non-cancer tissue. Malignant progression is associated with an epithelial-mesenchymal transition (EMT) program whereby epithelial cancer cells take on a mesenchymal phenotype and dissociate from a tumor mass, invade, and disseminate to distant metastatic sites. The objective of this study was to determine if epithelial and mesenchymal prostate cancer cells have different nuclear morphology. METHODS: Murine tibia injections of epithelial PC3 (PC3-Epi) and mesenchymal PC3 (PC3-EMT) prostate cancer cells were processed and stained with H&E. Cancer cell nuclear image data was obtained using commercially available image-processing software. Univariate and multivariate statistical analysis were used to compare the two phenotypes. Several non-parametric classifiers were constructed and permutation-tested at various training set fractions to ensure robustness of classification between PC3-Epi and PC3-EMT cells in vivo. RESULTS: PC3-Epi and PC3-EMT prostate cancer cells were separable at the single cell level in murine tibia injections on the basis of nuclear structure and texture remodeling associated with an EMT. Support vector machine and multinomial logistic regression models based on nuclear architecture features yielded AUC-ROC curves of 0.95 and 0.96, respectively, in separating PC3-Epi and PC3-EMT prostate cancer cells in vivo. CONCLUSIONS: Prostate cancer cells that have undergone an EMT demonstrated an altered nuclear structure. The association of nuclear changes and a mesenchymal phenotype demonstrates quantitative morphometric image analysis may be used to detect cancer cells that have undergone EMT. This morphometric measurement could provide valuable prognostic information in patients regarding the likelihood of [future] metastatic disease.