Cell Proliferation Status Research Articles

NT-proBNP as neuroendocrine tumor biomarker: beyond heart failure.

Neuroendocrine tumors (NETs) are rare neoplasms that occur in various locations throughout the body. Despite their usually benign character, they might manifest with distant metastases. N-terminal prohormone of brain natriuretic peptide (NT-proBNP) has previously been described as a useful biomarker in diagnosing carcinoid heart disease (CHD), a common advanced NETs manifestation. We observed plasma concentrations of NT-proBNP in metastatic midgut NETs over a 4-year period. We aimed to explore NT-proBNP concentrations in states of varying levels of cell proliferation and disease status. Our goal was to investigate NT-proBNP's role in predicting disease progression in relation to previous research and up-to-date scientific guidelines. We performed a retrospective multivariate analysis of NT-proBNP concentrations in 41 midgut NETs patients treated with somatostatin analogs, all with liver metastases. NT-proBNP concentrations were measured in every patient across 16 evenly distanced time points over a 48-month period and were compared to variables such as sex, age, grading, Ki-67, primary tumor location, and CT findings. NT-proBNP concentrations correlated positively with higher liver tumor burden, higher grading, high Ki-67 levels, and with progressive disease in CT. There were no differences in NT-proBNP levels with regard to primary location (ileum vs jejunum), sex, and age. We conclude that NT-proBNP is a useful analyte for monitoring NETs progression, due to its increased concentration in scenarios implying increased cellular proliferation. These long-term follow-up results align with previous findings and suggest an additional role for NT-proBNP in diagnostic algorithms, beyond a carcinoid heart disease biomarker.

The Role of cAMP-Dependent Intracellular Signaling Pathways in the Regulation of the Functions of Neural Stem Cells and Neuroglial Cells in Amyloid-β-Induced Neurodegeneration.

Under conditions of neurodegeneration modeled in vitro by the β-amyloid peptide-(25-35) fragment (Aβ25-35), we studied the role of individual links of cAMP-dependent intracellular signaling pathways in determining the proliferation and differentiation status of neural stem cells (NSCs) and colony-stimulating activity of supernatants from neuroglial cells. The important role of intracellular cAMP and PKA in the inhibition of the progression of the NSC cell cycle and stimulation of the process of their specialization induced by Aβ25-35 was found. The selective ability of PKA to block the production of factors constituting colony-stimulating activity by neuroglial cells under conditions of their cultivation in vitro with a neurotoxic agent was revealed. Our results suggests that inhibitors of adenylate cyclase and PKA can increase the degree of implementation of the growth potential of NSCs and conjugation of the processes of their proliferation and differentiation in Alzheimer's disease. At the same time, selective PKA blockers can also induce the production of NSC-stimulating factors by neuroglial cells.

Insights from a 30-year journey: function, regulation and therapeutic modulation of PD1.

PD1 was originally discovered in 1992 as a molecule associated with activation-induced cell death in T cells. Over the past 30 years, it was found that PD1 has a critical role in avoiding overactivation-induced cell death and autoimmunity, whereas its inhibition unleashes anticancer immunity. Here, we outline the journey from the discovery of PD1 to its role as a breakthrough target in cancer immunotherapy. We describe its regulation and function and examine how a mechanistic understanding of PD1 signalling suggests a central function in setting the T cell activation threshold, thereby controlling T cell proliferation, differentiation, exhaustion and metabolic status. This threshold theory, in combination with new insights into T cell metabolism and a better understanding of immune cell modulation by the microbiota, can provide guidance for the development of efficient combination therapies. Moreover, we discuss the mechanisms underlying immune-related adverse events after PD1-targeted therapy and their possible treatment.

Abstract 4741: NUDT21 regulates PIGN through APA mechanism to affect the progression of MDS and AML

Abstract Introduction: PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevent MDS leukemic transformation and progression. Alternative Polyadenylation (APA) regulation and 3' RNA cleavage are 2 known vital processes in cellular DNA damage response to maintain genomic stability. NUDT21 gene encodes protein that is one subunit of the cleavage factor Im complex required for 3' RNA cleavage and APA processing. Although PIGN gene has six potential polyadenylation sites, the exact role of NUDT21 product on PIGN expression regulation has not been illustrated before. Methods: At first, we analyzed NUDT21 gene expression profile in AML, MDS, and normal control specimens and to plot the relationship between NUDT21 gene expression status and patient survival length from 5 datasets. We also used correlation regression analysis method to predict the gene expression correlations between NUDT21 gene and PIGN gene. Then, we used Caspas9 technology and vector overexpression technology to knockout or overexpress PIGN in HEK293 cells; RNA interference to knockdown the NUDT21 gene in HEK293, HL60, K562, THP-1,SKM-1 and MUTZ-1 cells. MTT and CCK8 methods were used to evaluate cell proliferation. Meanwhile, cell cycle, apoptosis, Reactive Oxygen Species (ROS) and GPI-anchored proteins (CD55, CD59, and CD235) expression profiles were evaluated by flowcytometry; Immunofluorescence experiment, RT-PCR were used to evaluate the impact of NUDT21 gene knockdown on PIGN expression. in vivo mice siRNA interference tests were conducted to verify the impact of NUDT21 knockdown on PIGN gene expression. RNA-Seq & Dapars analyses were conducted on mRNAs from 3 cell lines with various PIGN gene expression status to observe the effects of NUDT21 knockdown on PIGN 3'UTR processing. Results: NUDT21 expression increases in AML and high-risk MDS. NUDT21 expression level is negatively correlated with overall survival. NUDT21 knockdown decreases cell proliferation but increases cell differentiation via prolonging G1 phase, decreasing S phase, and increasing apoptosis. NUDT21 knockdown increases cell membrane GPI-anchoring proteins (CD55, CD59, and CD235) expressions; increases ROS and Caspas3 expressions but decreases CyclinD1 expression. NUDT21 knockout can increase PIGN expression, but PIGN expression status has no impact on NUDT21 expression. RT-PCR and WB results showed that NUDT21 knockdown can increase TP53 expression but decrease p21 expression. in vivo mice siRNA interference tests confirmed that NUDT21 knockdown increases PIGN gene expression. The RNA-Seq/Dapars analyses results showed that 3'UTR of PIGN transcript was significantly shortened and PIGN expression increased after NUDT21 knockdown. Conclusion: NUDT21 modulates PIGN gene expression through APA mechanism. PIGN polyadenylation may regulate cell proliferation status via modulating cell cycle and apoptosis. Citation Format: Jeffrey J. Pu, Fanggang Ren, Na Zhang, Shuo Li, Lan Zhang, Eric Miller, Guirong Wang, Hongwei Wang. NUDT21 regulates PIGN through APA mechanism to affect the progression of MDS and AML. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4741.

Abstract IA014: The role of aging in reactivation from metastatic melanoma dormancy

Abstract Disseminated cancer cells (DCCs) that escape the primary site can seed in distal tissues, but may take several years, or even decades to grow out into overt metastases, a phenomenon termed tumor dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully model or characterize dormancy within melanoma. Our previous data has indicated that while tumors grow more slowly in aged mouse skin, they form more aged lung metastases. We have previously shown that dermal fibroblasts were key drivers of age-related changes in the skin to promote a more aggressive melanoma phenotype. A model of melanoma progression that addresses these microenvironmental complexities is the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state. We queried whether the lung microenvironment undergoes similar age-related changes that provide a permissive niche for metastatic outgrowth. Our new data identified that while Wnt5A and AXL are required for efficient dissemination from the primary tumor towards metastatic tissues, they act as master regulators of activating melanoma dormancy within the lung. The young lung microenvironment maintains this dormant melanoma state; however, age induced reprograming of lung fibroblasts increases their secretion of the soluble Wnt antagonist sFRP1, which inhibits both Wnt5A and AXL to enable reactivation from dormancy and the formation of larger metastatic colonies in the aged lung. We find that downregulation of AXL in melanoma cells leads to an increase in the related TAM family receptor MERTK, which promotes a proliferative melanoma phenotype, enables reactivation from dormancy and induces efficient metastatic outgrowth in aged models. Follow up data from these models highlights decreases in immune infiltration of CD4+ and CD8+ T-cells, which appears to be partially mediated by an increase in secretion of Arginase-1 (ARG1) from aged lung fibroblasts. Further, we find that aged lung fibroblasts secrete CXCL1 which promotes infiltration of immunosuppressive T-regulatory cells and Myeloid Derived Suppressive Cells. Finally, we see dramatic changes in extracellular matrix secretion and remodeling from young vs aged lung fibroblasts, which alters in vitro melanoma growth to promote dormancy in melanoma cells grown on young lung fibroblast ECM. Overall, we find that a plethora of age-related changes in metastatic microenvironments such as the lung contribute to an increase in melanoma progression relative to younger niches. This not only provides novel tools to assess metastatic dormancy in melanoma, which is critically understudied due to a lack of pre-clincal in vitro and in vivo models, but also necessitates the idea that age needs to be considered in pre-clinical modelling of age-related cancers such as melanoma, where the median age of diagnosis is 63, and when there is a clear increase in incidence, mortality and metastasis within geriatric patients. Citation Format: Mitchell Fane, Yash Chhabra, Gretchen Alicea, Daniel Zabransky, Laura Hüser, Devin Maranto, Julio Aguirre-Ghiso, Ashani Weeraratna. The role of aging in reactivation from metastatic melanoma dormancy [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA014.

Association between proliferation status of infected and non-infected mononuclear cells with tissue lesions in acute bovine theileriosis.

Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tams1 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes.

Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small Intestinal Organoids Using Cholera Toxin.

Live cell microscopies of in vitro, ex vivo, and in vivo experimental intestinal models enable visualizing cell proliferation, differentiation, and functional cellular status in response to intrinsic and extrinsic (e.g., in the presence of microbiota) factors. While the use of transgenic animal models expressing biosensor fluorescent proteins can be laborious and not compatible with clinical samples and patient-derived organoids, the use of fluorescent dye tracers is an attractive alternative. In this protocol, we describe how the differentiation-dependent intestinal cell membrane composition can be labeled using fluorescent cholera toxin subunit B (CTX) derivatives. By using the culture of mouse adult stem cell-derived smallintestinal organoids, we show that CTX can bind specific plasma membrane domains in differentiation-dependent manner. Green (Alexa Fluor 488) and red (Alexa Fluor 555) fluorescent CTX derivatives also display additional contrast in a fluorescence lifetime domain, when probed by the fluorescence lifetime imaging microscopy (FLIM), and can be used together with other fluorescent dyes and cell tracers. Importantly, CTX staining remains confined to specific regions in the organoids after fixation, which enables using it in both live cell and fixed tissue immunofluorescence microscopies.

CFIm-mediated alternative polyadenylation safeguards the development of mammalian pre-implantation embryos.

Alternative polyadenylation (APA) gives rise to transcripts with distinct 3' untranslated regions (3' UTRs), thereby affecting the fate of mRNAs. APA is strongly associated with cell proliferation and differentiation status, and thus likely plays a critical role in the embryo development. However, the pattern of APA in mammalian early embryos is still unknown. Here, we analyzed the 3' UTR lengths in human and mouse pre-implantation embryos using available single cell RNA-seq datasets and explored the underlying mechanism driving the changes. Although human and mouse early embryos displayed distinct patterns of 3' UTR changing, RNA metabolism pathways were involved in both species. The 3' UTR lengths are likely determined by the abundance of the cleavage factor I complex (CFIm) components NUDT21 and CPSF6 in the nucleus. Importantly, depletion of either component resulted in early embryo development arrest and 3' UTR shortening. Collectively, these data highlight an essential role for APA in the development of mammalian early embryos.

Comparative analysis of mesenchymal stem/stromal cells derived from human induced pluripotent stem cells and the cognate umbilical cord mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSCs) show tremendous potential for regenerative medicine due to their self-renewal, multi-differentiation and immunomodulatory capabilities. Largely studies had indicated conventional tissue-derived MSCs have considerable limited expandability and donor variability which hinders further application. Induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) have created exciting source for standardized cellular therapy. However, the cellular and molecular differences between iMSCs and the cognate tissue-derived MSCs remains poorly explored. In this study, we first successfully reprogrammed human umbilical cords-derived mesenchymal stem/stromal cells (UMSCs) into iPSCs by using the cocktails of mRNA. Subsequently, iPSCs were further differentiated into iMSCs in xeno-free induction medium. Then, iMSCs were compared with the donor matched UMSCs by assessing proliferative state, differentiation capability, immunomodulatory potential through immunohistochemical analysis, flow cytometric analysis, transcriptome sequencing analysis, and combine with coculture with immune cell population. The results showed that iMSCs exhibited high expression of MSCs positive-makers CD73, CD90, CD105 and lack expression of negative-maker cocktails CD34, CD45, CD11b, CD19, HLA-DR; also successfully differentiated into osteocytes, chondrocytes and adipocytes. Further, the iMSCs were similar with their parental UMSCs in cell proliferative state detected by the CCK-8 assay, and in cell rejuvenation state assessed by β-Galactosidase staining and telomerase activity related mRNA and protein analysis. However, iMSCs exhibited similarity to resident MSCs in Homeobox (Hox) genes expression profile and presented better neural differentiation potential by activation of NESTIN related pathway. Moreover, iMSCs owned enhanced immunosuppression capacity through downregulation pools of pro-inflammatory factors, including IL6, IL1B etc. and upregulation anti-inflammatory factors NOS1, TGFB etc. signals. In summary, our study provides an attractive cell source for basic research and offers fundamental biological insight of iMSCs-based therapy.

Visualization of hypoxia in cancer cells from effusions in animals and cancer patients.

Tumor hypoxia is frequently observed in primary solid malignancies, but the hypoxic status of tumor cells floating in body cavity effusions is largely unknown, especially in patients. This study was to observe the hypoxia and proliferation status of cancer cells floating in effusions in mice and patients. The distribution of hypoxia in cancer cells floating in ascites was first studied in nude mice. Hypoxia was detected by immunofluorescent visualization of pimonidazole and GLUT-1. For cancer patients, we retrospectively collected 21 ascites and 7 pleural effusion sample blocks of cancer patients, which were confirmed to contain tumor cells. Immunohistochemistry was performed to detect the expression of endogenous hypoxic markers HIF-1α and GLUT-1, proliferation index Ki-67. 18F-FDG PET/CT was performed to detect the glucose metabolism status of tumor cells in effusions. The tumor cells collected from ascites were positive for pimonidazole and GLUT-1, which suggesting that the cancer cells floating in ascites were hypoxic. Patterns of tumor hypoxia in human patients are similar to those observed in animal. HIF-1α and GLUT-1 were expressed by tumor cells in nearly all 28 cytological cases. For Ki-67 index, ascites tumor cells had a relatively low expression level compared with their corresponding primary or its metastatic lesions. Tumor cells in effusions showed high 18F-FDG uptake indicated the enhanced activity of glucose metabolism. Tumor cells in body cavity effusions, as a unique subgroup of tumor, are in a state of hypoxia and low proliferation, which would be one of the driven causes of chemo-radiotherapy resistance. Novel therapeutic interventions are urgently needed to overcome tumor hypoxia.

MBNL1 controls cardiomyocyte differentiated versus proliferative cell state

MBNL1 controls cardiomyocyte differentiated versus proliferative cell state

PATH-30. BIOLOGIC AND CLINICAL SIGNIFICANCE OF NEOPLASTIC AND IMMUNE CELL STATES IN GROUP3 AND GROUP 4 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a heterogeneous malignant childhood brain cancer, classified into four molecular subgroups, WNT, SHH, Group3, and Group4, based on transcriptional, genetic, and clinical features. We aimed to understand the tumor microenvironment of Group 3 and Group 4 tumors utilizing single-cell transcriptome data. We identified four neoplastic cell states, including stemness (TNF-α), proliferative, mesenchymal and quiescence, and 11 broad lymphoid and myeloid cell types from single-cell transcriptomes. We applied CIBERSORTx to enumerate cell type proportion and CODEFACS on 73 bulk MB RNAseq profiles to determine cell type-specific abundance and gene expression profiles. We noted significant associations (both positive and negative) between the abundance of neoplastic cell states and microenvironmental cell types in both group3 and group4 MB. For example, the invasive cell state negatively correlated with dendritic cells in Group3 and Group4 tumors, but the invasive cell state was oppositely correlated with a macrophage subset in group 3 versus group 4 tumors, Survival analysis showed a significant association of higher proliferative cell state with poorer survival in Group3 MB while in group4 elevated invasive cell state, as well as elevated α, NK, and CD4-T cell abundance showed trends for poorer prognosis. Using Ecotyper, we determined cellular ecotypes based on cell type-specific transcriptome states. An ecotype containing B cell, hematopoietic, dendritic, and proliferative cell states was associated with poor prognosis in group3 and group4 tumors. We further analyzed ligand-receptor interaction between cell type states of poor prognosis ecotype; and found several interactions between ligands from immune cells (SEMA4G, LAMB2) and receptors on neoplastic cells (PLXNB2). Conversely, extracellular matrix ligands on neoplastic cells (COL1A1/2 and FN1) were found on this analysis potentially associated with integrin receptors on immune cells. Overall, our study highlights the potential biologic and clinical significance of neoplastic and immune cell states in group3/4 MB.

Osteoblast Secretome Modulated by Abiraterone Treatment Affects Castration Resistant Prostate Cancer Cell Proliferation.

Abiraterone is a selective inhibitor of androgen biosynthesis approved for the treatment of metastatic patients affected by castration-resistant or castration-sensitive prostate cancer. Intriguingly, clinical data revealed that abiraterone also delayed disease progression in bone improving bone-related endpoints. Our group has previously demonstrated in vitro a direct effect of abiraterone on osteoclast and osteoblast function suggesting its ability to modulate bone microenvironment. Here, we performed an extensive proteomic analysis to investigate how abiraterone influences osteoblast cell secretome and, consequently, osteoblast/prostate cancer cells interaction. A panel of 507 soluble molecules were analyzed in osteoblast conditioned media (OCM) obtained from osteoblast treated or not with abiraterone. Subsequently, OCM was added to prostate cancer cells to investigate its potential effect on prostate cancer cell proliferation and androgen receptor (AR) activation status. Out of 507 screened molecules, 39 of them were differentially expressed in OCM from osteoblasts treated with abiraterone (OCM ABI) compared to OCM obtained from untreated OBs (OCM CTRL). Pathway enrichment analysis revealed that abiraterone down-modulated the release of specific osteoblast soluble factors, positively associated with cell proliferation pathways (false discovery rate adjusted p-value = 0.0019). In vitro validation data showed that OCM ABI treatment significantly reduced cancer proliferation in C4-2B cells (p = 0.022), but not in AR- negative PC-3 cells. Moreover, we also found a reduction in AR activation in C4-2B cells (p = 0.017) confirming the “indirect” anti-tumor AR-dependent effect of abiraterone mediated by osteoblasts. This study provides the first evidence of an additional antitumor effect of abiraterone through the modulation of multiple osteoblast proliferative signals.

Abstract SY24-04: And by a sleep to say we end: Tumor dormancy, aging, and recurrent disease

Abstract Disseminated cancer cells (DCCs) that escape the primary site can seed in distal tissues, but may take several years, or even decades to grow out into overt metastases, a phenomenon termed tumor dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully model or characterize dormancy within melanoma. Here, we show that age-related changes in the lung microenvironment facilitate a permissive niche for efficient outgrowth of disseminated dormant tumor cells, in contrast to the aged skin, where age-related changes suppress melanoma growth but drive dissemination. A model of melanoma progression that addresses these microenvironmental complexities is the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state. We have previously shown that dermal fibroblasts are key orchestrators of promoting phenotype switching in primary melanoma tumors via changes in the secretion of soluble factors during aging. Our new data identify Wnt5A as a master regulator of activating melanoma DCC dormancy within the lung, which initially enables efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of soluble Wnt factors enabling efficient metastatic outgrowth. Overall, we find that age-induced changes in distal metastatic microenvironments promotes efficient reactivation of dormant melanoma cells in the lung. Further these changes are also reflected in the response of tumor cells to therapy, linking dormancy to recurrent disease. Citation Format: Ashani T. Weeraratna. And by a sleep to say we end: Tumor dormancy, aging, and recurrent disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-04.

Stromal changes in the aged lung induce an emergence from melanoma dormancy.

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5Ain melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axiswithin melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.

Selection of RNA-based evaluation methods for tumor microenvironment by comparing with histochemical and flow cytometric analyses in gastric cancer

Understanding the tumor microenvironment (TME) and anti-tumor immune responses in gastric cancer are required for precision immune-oncology. Taking advantage of next-generation sequencing technology, the feasibility and reliability of transcriptome-based TME analysis were investigated. TME of 30 surgically resected gastric cancer tissues was analyzed by RNA-Seq, immunohistochemistry (IHC) and flow cytometry (FCM). RNA-Seq of bulk gastric cancer tissues was computationally analyzed to evaluate TME. Computationally analyzed immune cell composition was validated by comparison with cell densities established by IHC and FCM from the same tumor tissue. Immune cell infiltration and cellular function were also validated with IHC and FCM. Cell proliferation and cell death in the tumor as assessed by RNA-Seq and IHC were compared. Computational tools and gene set analysis for quantifying CD8+ T cells, regulatory T cells and B cells, T cell infiltration and functional status, and cell proliferation and cell death status yielded an excellent correlation with IHC and FCM data. Using these validated transcriptome-based analyses, the immunological status of gastric cancer could be classified into immune-rich and immune-poor subtypes. Transcriptome-based TME analysis is feasible and is valuable for further understanding the immunological status of gastric cancer.

High-Concentration Metformin Reduces Oxidative Stress Injury and Inhibits the Growth and Migration of Clear Cell Renal Cell Carcinoma.

Objective To explore the mechanism of metformin in treating CCRCC. Methods Prospective cohort study was conducted. SOD and cyclin D in six CCRCC samples donated by volunteers were detected to compare the degree of oxidative stress injury and the status of cell proliferation. 786-0 CCRCC cells were cultured in vitro with different concentrations of metformin, and MTT assay and Transwell cell migration and wound healing assay were used to detect their proliferation and migration. After culture, SOD and cyclin D in 786-0 CCRCC cells were also detected. Results In the edge tissue, SOD was lower than in the tumor nest and normal tissue, and cyclin D was highly expressed. In grade II CCRCC, SOD was higher than in grade IV CCRCC, but cyclin D was also highly expressed in grade IV CCRCC. The cell proliferation rate and density of the metformin group were lower than the control group, while in the high-concentration metformin group, it was lower than medium- and low-concentration groups. After culture, the migration of 786-0 cells in the metformin group was significantly lower than that in the control group, the wound healing rate was decreased, and the migration and wound healing rates in the high-concentration metformin group were significantly lower than those in the medium- and low-concentration groups. However, the SOD of the metformin group was higher than the control group, but the cyclin D was lower, while the SOD was higher than medium- and low-concentration groups in the high-concentration group, but the cyclin D was lower after cultured. Conclusion High-concentration metformin can reduce oxidative stress injury, increase the expression of SOD in CCRCC, and reduce cyclin D in CCRCC to inhibit proliferation and migration, which has optimistic prospects and application value in controlling the progression of CCRCC.

Examination of the effects of microRNA-145-5p and phosphoserine aminotransferase 1 in colon cancer

ABSTRACT Previous studies manifested that microRNA-145-5p is pivotal in the development of various cancers. Nevertheless, the potential function of microRNA-145-5p in colorectal cancer remains unclear. This study attempted to investigate the potential role and possible mechanism of microRNA-145-5p in colon cancer. MicroRNA-145-5p and phosphoserine aminotransferase 1 (PSAT1) levels in colon cancer cells were assayed via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation and cell cycle status were assessed using Cell Counting Kit-8, colony formation, and flow cytometry. The target binding relationship of microRNA-145-5p and PSAT1 was identified using bioinformatics analysis and dual-luciferase reporter gene assay. The result of qRT-PCR disclosed that microRNA-145-5p was markedly down-regulated and PSAT1 level was up-regulated in colon cancer cell lines. Besides, enforced microRNA-145-5p level repressed proliferation of colon cancer cells, and cells were arrested in G0-G1 phase. Bioinformatics analysis and dual-luciferase reporter genes confirmed that PSAT1 was a downstream target of microRNA-145-5p. Enforced PSAT1 level remarkably modulated cell cycle and fostered cell proliferation. Furthermore, rescue experiments displayed that microRNA-145-5p restrained cell cycle progression and cell proliferation and forced PSAT1 level could partially reverse this process. Taken together, our findings demonstrated that microRNA-145-5p repressed colon cancer cell cycle progression and cell proliferation via targeting PSAT1. Our findings identified microRNA-145-5p as an essential tumor repressor gene in colon cancer and may provide a novel biomarker for colon cancer.

Increased Neutrophil Infiltration and Epithelial Cell Proliferation in Sinonasal Inverted Papilloma Compared to Contralateral Nasal Polyps.

Sinonasal inverted papilloma (IP) is a rare and benign epithelial tumor in the sinonasal tract. Recent study suggested the potential role of chronic inflammation in the pathogenesis of IP. This study aims to compare the inflammatory pattern, the capacity of epithelial cell proliferation and EGFR mutation status of unilateral IP with contralateral polyp tissue. Sixteen patients with unilateral IP and contralateral nasal polyps (NP) were identified through a retrospective chart review. The neutrophil and eosinophil infiltration in IP and NP were assessed by immunostaining for neutrophil elastase and major basic protein (MBP). Immunohistochemistry was also used to assess the expression of FoxM1, Ki67 and cyclin D1 in IP tissue and contralateral NP. Sanger sequencing was used to evaluate the EGFR mutations. The neutrophil count in IP was significantly higher than contralateral NP and 68.8% patients presented with neutrophilic inflammation, whereas only 37.5% contralateral NP tissue showed neutrophilic inflammation. The percentage of positive FoxM1-staining cells was significantly increased in IP, and positively correlated with the percentage of cells with positive staining for cyclin D1 and ki67 as well as neutrophil counts. EGFR exon 20 insertions were detected in 14 (87.5%) IP samples and no EGFR mutations were found in contralateral NP sample. Our study demonstrated distinct inflammatory pattern between IP and contralateral NP and implied the oncogenic role of neutrophils in the pathogenesis of IP. EGFR mutations may be the early event to initiate IP development by enhancing epithelial cell proliferation.

Predicting the Proliferation of Tongue Cancer With Artificial Intelligence in Contrast-Enhanced CT.

Tongue squamous cell carcinoma (TSCC) is the most common oral malignancy. The proliferation status of tumor cells as indicated with the Ki-67 index has great impact on tumor microenvironment, therapeutic strategy making, and patients’ prognosis. However, the most commonly used method to obtain the proliferation status is through biopsy or surgical immunohistochemical staining. Noninvasive method before operation remains a challenge. Hence, in this study, we aimed to validate a novel method to predict the proliferation status of TSCC using contrast-enhanced CT (CECT) based on artificial intelligence (AI). CECT images of the lesion area from 179 TSCC patients were analyzed using a convolutional neural network (CNN). Patients were divided into a high proliferation status group and a low proliferation status group according to the Ki-67 index of patients with the median 20% as cutoff. The model was trained and then the test set was automatically classified. Results of the test set showed an accuracy of 65.38% and an AUC of 0.7172, suggesting that the majority of samples were classified correctly and the model was stable. Our study provided a possibility of predicting the proliferation status of TSCC using AI in CECT noninvasively before operation.