Single-cell and spatial analyses reveal the association between gene expression of glutamine synthetase with the immunosuppressive phenotype of APOE+CTSZ+TAM in cancers.

An immunosuppressive state is regulated by various factors in the tumor microenvironment (TME), including, but not limited to, metabolic plasticity of immunosuppressive cells and cytokines secreted by these cells. We used single-cell RNA-sequencing (scRNA-seq) data and applied single-cell flux estimation analysis to characterize the link between metabolism and cellular function within the hypoxic TME of colorectal (CRC) and lung cancer. In terms of metabolic heterogeneity, we found myeloid cells potentially inclined to accumulate glutamine but tumor cells inclined to accumulate glutamate. In particular, we uncovered a tumor-associated macrophage (TAM) subpopulation, APOE+CTSZ+TAM, that was present in high proportions in tumor samples and exhibited immunosuppressive characteristics through upregulating the expression of anti-inflammatory genes. The proportion of APOE+CTSZ+TAM and regulatory T cells (Treg) were positively correlated across CRC scRNA-seq samples. APOE+CTSZ+TAM potentially interacted with Treg via CXCL16-CCR6 signals, as seen by ligand-receptor interactions analysis. Notably, glutamate-to-glutamine metabolic flux score and glutamine synthetase (GLUL) expression were uniquely higher in APOE+CTSZ+TAM, compared with other cell types within the TME. GLUL expression in macrophages was positively correlated with anti-inflammatory score and was higher in high-grade and invasive tumor samples. Moreover, spatial transcriptome and multiplex immunofluorescence staining of samples showed that APOE+CTSZ+TAM and Treg potentially colocalized in the tissue sections from CRC clinical samples. These results highlight the specific role and metabolic characteristic of the APOE+CTSZ+TAM subpopulation and provide a new perspective for macrophage subcluster-targeted therapeutic interventions or metabolic checkpoint-based cancer therapies.

Extended transcriptome analysis reveals genome-wide lncRNA-mediated epigenetic dysregulation in colorectal cancer.

It is estimated that the rate of epigenetic changes may be orders of magnitude higher than that of genetic changes and that purely epigenetic mechanisms may explain why cancers arise with few or no recurrent mutations. However, supporting evidence remains limited, partly due to the cost of experimentally studying genome-wide epigenetic dysregulation. Since genome modification enzymes are recruited by long noncoding RNAs (lncRNAs) to specific genomic sites, analyzing differentially expressed genes and differentially methylated regions (DMRs) at the DNA binding sites of differentially expressed lncRNAs is important for uncovering epigenetic dysregulation. We performed RNA-seq and MeDIP-seq on a set of colorectal cancer (CRC) and normal colon samples and developed an analysis pipeline for combined analyses of gene expression, DNA methylation, and lncRNA/DNA binding. The genes identified in our data and important for CRC agree with widely reported findings. We found that aberrantly transcribed noncoding transcripts may epigenetically dysregulate genes, that correlated gene expression is significantly determined by epigenetic dysregulation, that differentially expressed noncoding transcripts and their epigenetic targets form distinct modules in different cancer cells, and that many hub lncRNAs in these modules are primate-specific. These results suggest that lncRNA-mediated epigenetic dysregulation greatly determines aberrant gene expression and that epigenetic dysregulation is highly species-specific. The analysis pipeline can effectively unveil cancer- and cell-specific modules of epigenetic dysregulation, and such modules may provide novel clues for identifying diagnostic, therapeutic, and prognostic targets for epigenetic dysregulation.

The targeted transduction of MMP-overexpressing tumor cells by ACPP-HPMA copolymer-coated adenovirus conjugates.

We have designed and tested a new way to selectively deliver HPMA polymer-coated adenovirus type 5 (Ad5) particles into matrix metalloproteinase (MMP)-overexpressing tumor cells. An activatable cell penetrating peptide (ACPP) was designed and attached to the reactive 4-nitrophenoxy groups of HPMA polymers by the C-terminal amino acid (asparagine, N). ACPPs are activatable cell penetrating peptides (CPPs) with a linker between polycationic and polyanionic domains, and MMP-mediated cleavage releases the CPP portion and its attached cargo to enable cell entry. Our data indicate that the transport of these HPMA polymer conjugates by a single ACPP molecule to the cytoplasm occurs via a nonendocytotic and concentration-independent process. The uptake was observed to finish within 20 minutes by inverted fluorescence microscopy. In contrast, HPMA polymer-coated Ad5 without ACPPs was internalized solely by endocytosis. The optimal formulation was not affected by the presence of Ad5 neutralizing antibodies during transduction, and ACPP/polymer-coated Ad5 also retained high targeting capability to several MMP-overexpressing tumor cell types. For the first time, ACPP-mediated cytoplasmic delivery of polymer-bound Ad5 to MMP-overexpressing tumor cells was demonstrated. These findings are significant, as they demonstrate the use of a polymer-based system for the targeted delivery into MMP-overexpressing solid tumors and highlight how to overcome major cellular obstacles to achieve intracellular macromolecular delivery.

Influence of cervical spine position, turning time, and cervical segment on cadaver intradiscal pressure during cervical spinal manipulative therapy.

OBJECTIVE: The purpose of this study was to determine influences of cervical spine positions, turning times, and cervical segments on cadaver intradiscal pressure (IDP) during cervical spinal manipulative therapy (SMT). METHODS: We simulated cervical SMT with stretching and rotation on 7 fresh adult cadaver specimens in the material test system machine. The changes in IDP for cervical intervertebral disks (C3/4, C4/5, and C5/6) during 4 different stages of cervical SMT (physiologic state, end of the traction stage, turning stage, and finish time) were monitored. Five different cervical positions (extension 20°, extension 10°, neutral position, flexion 10°, flexion 20°) and 3 different turning times (0.06, 0.11, 0.16 second) of IDP were monitored, using micropressure sensors. RESULTS: The variable tendency of cervical IDP presents a "V"-shaped curve during SMT. The 4 stages of SMT had significantly different IDP (F=5498.956; P<.001). There were also significant differences in IDP between 5 cervical positions ([F=1371.216; P<.001], [flexion 20°>flexion 10°>neutral position>extension 10°>extension 20°]), 3 turning times ([F=419.530; P<.001], [0.06>0.11>0.16 seconds]), and 3 cervical segments ([F=84.282; P<.001], [C3/4

[Infantile DiGeorge syndrome: autopsy diagnosis and clinicopathologic analysis in 5 cases].

OBJECTIVE: To investigate clinicopathological features of DiGeorge syndrome (DGS). METHOD: The clinical features, histological and immunohistochemical findings were analyzed in 5 cases of DGS by autopsy. RESULTS: Five cases of DGS in male infants aged 4 days, 1 month, 7 months, 10 months, and 13 months respectively. Gross and microscopic observations revealed that thymic cortex was depleted of lymphocytes or showed few, dispersed lymphocytes. The thymic medulla showed predominantly epithelial cells with calcified Hassall bodies as well as lymphocyte depletion. T lymphocytes were also scarce in the tonsils, lymph nodes, spleen, and mucosa-associated lymphatic tissue of ileum. In addition, 3 of the 5 patients also showed parathyroid aplasia or dysplasia, and congenital hypertrophy of the ventricular septum. CONCLUSIONS: The pathological changes indicate that clinicians should be aware of defects of immune system if the infants suffer from severe infections. Pathologists should recognize the importance of abnormalities of lymphohematopoietic tissues in the diagnosis of primary immunodeficiency diseases such as DGS.

A five-gene signature as a potential predictor of metastasis and survival in colorectal cancer.

To understand the molecular mechanisms of metastasis and prognosis of colorectal cancer (CRC), we isolated single cell-derived progenies (SCPs) from SW480 cells in vitro and compared their metastatic potential in an orthotopic CRC tumour model in vivo. Two groups of SCPs with the capability of high and low metastasis, respectively, were obtained. By analysing the gene expression profiles of high (SCP51), low (SCP58) metastatic SCPs, and their parental cell line (SW480/EGFP), we demonstrated that 143 genes were differentially expressed either between SCP51 and SCP58 or between SCP58 and SW480/EGFP. Gene-annotation enrichment analysis of DAVID revealed 80 genes in the top ten clusters of the analysis (gene enrichment score > 1). Of the 80-gene set, 32 genes are potentially involved in metastasis, as revealed by Geneclip. Five putative metastatic genes (LYN, SDCBP, MAP4K4, DKK1, and MID1) were selected for further validations. Immunohistochemical analysis in a cohort of 181 CRC clinical samples showed that the individual expression of LYN, MAP4K4, and MID1, as well as the five-gene signature, was closely correlated with lymph node metastasis in CRC patients. More importantly, the individual expression of LYN, MAP4K4, SDCBP, and MID1, as well as the five-gene signature, was significantly correlated with overall survival in CRC patients. Thus, our five-gene signature may be able to predict metastasis and survival of CRC in the clinic, and opens new perspectives on the biology of CRC.

Focal adhesion plaque associated cytoskeletons are involved in the invasion and metastasis of human colorectal carcinoma.

The protein and mRNA expression of focal adhesion plaque associated cytoskeletons, including talin, vinculin, paxillin, and tensin, was studied using immunofluorescence in combination with confocal laser scanning microscopy and fluorescent quantitative polymerase chain reaction in 41 matched samples of human normal colorectal mucosae, primary colorectal adenocarcinomas, and 19 separate lymph node metastases. All specimens showed expression. The results showed talin, vinculin, tensin, and paxillin expression were correlated with carcinogenesis, invasion, and metastasis of colorectal carcinoma (CRC). Talin, vinculin, and tensin underwent downregulation while paxillin went up. So these cytoskeletons may play bidirectional regulating roles during the progression of CRC.

[Overexpression of Tiam1 gene and its relationship with invasive and metastatic ability of nasopharyngeal carcinoma].

OBJECTIVE: To explore biological aspects of Tiam1 gene expression in nasopharyngeal carcinoma cells. METHODS: Tiam1/C1199HA expression plasmids were transfected into nasopharyngeal carcinoma cells of C666-1 and CNE1 by lipofectamine2000. RT-PCR, real-time PCR and Western blot Analyses were performed to evaluate the expression of Tiam1 mRNA and protein levels, respectively. In vitro cell adhesion, wound healing and matrigel invasion assays were used to study the biological impact of Tiam1 on cell adhesion, mobility and invasion. RESULTS: Tiam1 over expression significantly increased the abilities of adhesion, migratory and invasion of C666-1 and CNE1 cells, comparing with that of the control untransfected cells (P < 0.05). CONCLUSION: Tiam1 expression correlates with the invasion and metastasis of nasopharyngeal carcinoma cells.

Down-regulated expression of SATB2 is associated with metastasis and poor prognosis in colorectal cancer.

To identify novel biomarkers of metastasis of colorectal cancer (CRC), we developed an orthotopic implantation model of murine CRC and selected in vivo M5, a subclone of the SW480 CRC cell line with enhanced potential for metastasis to the liver. We compared the differences in the gene expression profiles between M5 and SW480 cells using gene expression profiling. We found that expression of special AT-rich sequence-binding protein 2 (SATB2) was down-regulated in M5 cells. Immunohistochemical analysis of 146 colorectal tumour samples showed that underexpression of SATB2 was strongly correlated with poor prognosis, tumour invasion, lymph node metastasis, distant metastasis, and Dukes' classification for CRC. Univariate and multivariate survival analyses further showed that SATB2 expression was a potential favourable prognostic factor for CRC. These results demonstrated not only that SATB2 is a potential novel prognostic factor for CRC, but also that selection of a highly metastatic clone of SW480 in vivo coupled with gene expression profiling is a powerful approach to identifying prognostic markers for CRC.

Over-expression of CDH22 is associated with tumor progression in colorectal cancer.

CDH22, a member of the cadherins family, is highly expressed in the pituitary gland and the brain. It has been previously found that CDH22 is involved in morphogenesis and tissue formation in neural and non-neural cells of the brain and neuroendocrine organ. However, little is known about its role in colorectal cancer. Here the role of CDH22 in colorectal tumor was tested, and effects of inhibition of CDH22 expression on proliferation and metastasis were examined. We found that CDH22 was over-expressed at both transcriptional and translational levels in colorectal cancer and lymphatic metastasis of colorectal carcinoma, compared with normal colorectal mucosa. CDH22 over-expression was significantly associated with invasion and metastasis of colorectal cancer both at protein and mRNA levels. CDH22 knockdown partially blocked proliferation of colorectal cancer cells in vivo and in vitro. CDH22 knockdown was sufficient to attenuate invasion of colorectal cancer cells and inhibit tumor metastasis in a mouse model of colon surgical orthotopic implantation. Our results reveal for the first time a new role of CDH22 in progression of colorectal cancer.

PRL-3 promotes epithelial mesenchymal transition by regulating cadherin directly.

PRL-3 is a key gene associated with progression and metastasis of colorectal cancer. Recently PRL-3 was suggested to promote epithelial mesenchymal transition (EMT) by downregulating E-cadherin expression. But the mechanisms of EMT induced by PRL-3 remain largely unknown. Here we found that PRL-3 could also promote EMT in a colorectal cancer cell model SW480 with deficient E-cadherin expression in vivo and in vitro. PRL-3 stable overexpression or knockdown SW480 cells were injected subcutaneously into nude mice. Immunohistochemical analyses of tumor samples from nude mice showed that PRL-3 promoted upregulation of mesenchymal marker vimentin and downregulation of epithelial markers E-cadherin and cytokeratin. Glycogen synthase kinase-3beta inactivated by PRL-3 as assessed by phosphospecific antibodies was a key event in EMT induced by PRL-3. Inhibition of glycogen synthase kinase-3beta by lithium chloride, a highly selective inhibitor, leading to phosphorylation of glycogen synthase kinase-3beta increased Snail expression. In order to identify the direct effects of PRL-3, we isolated CDH22, one member of cadherin family, as a new candidate of interacting proteins of PRL-3 in yeast two-hybrid systems, and the interaction was confirmed in vitro by GST pull-down assay or in exogenous cell systems and endogenous colorectal cancer cells by co-immunoprecipitation assay and co-localization analysis. We observed that PRL-3 promoted downregulation of CDH22 expression. Interestingly, expression of E-cadherin was recovered in SW480 cells after PRL-3 was knocked-down. Our results first linked PRL-3 to cadherin directly. It provided new insights into the regulatory mechanisms of EMT induced by PRL-3.

[Relationship between Tiam-1 expression and the biological behaviors of nasopharyngeal carcinoma].

OBJECTIVE: To investigate the relationship between the protein expression of T-lymphoma invasion and metastasis gene 1 (Tiam-1) and the biological behaviors of nasopharyngeal carcinoma (NPC). METHODS: Immunohistochemistry was performed to detect the expressions of Tiam-1 protein in 60 specimens of NPC tissue, 20 specimens of chronic nasopharyngitis (CN) tissue, and 6 tumor tissues from nude mice inoculated with metastatic human NPC cells. RESULTS: The positivity rate and average score for Tiam-1 expression were significantly higher in NPC tissues than in CN tissue (63.33% vs 36.67%, 2.9167 +/- 1.3057 vs 0.7000 +/- 0.9234; chi(2)=20.429, P=0.001; t=7.0162, P=0.0000, respectively). No difference was found in Tiam-1 expression among NPC patients in different T stages (F=2.36, P=0.0811), while the expression differed significantly between the patients with lymph node metastasis and those without metastasis, and also between patients with organ metastasis and those without (P=0.0001). High Tiam-1 expressions were found in the tumor tissues in nude mice inoculated with metastatic NPC cells. CONCLUSION: Tiam-1 expression is closely associated with the invasiveness and metastasis of NPC, indicating that Tiam-1 is an important factor that promotes the invasion and metastasis of NPC.

Overexpression of Tiam1 in hepatocellular carcinomas predicts poor prognosis of HCC patients.

Little research has been done to test the usefulness of T-lymphoma invasion and metastasis 1 (Tiam1) as a prognostic marker for hepatocellular carcinoma (HCC). In this study, we investigated Tiam1 expression and its prognostic value for HCC. HCC surgical tissue samples were taken from 152 HCC patients who had been followed up for 5 years. Overexpression of Tiam1 (Tiam1 2+ to 3+) was shown in 63.8% of the cases. The Tiam1 expression level did not correlate with any clinicopathological parameters. However, survival analysis showed that the Tiam1 overexpression group had a significantly shorter overall survival time than the Tiam1 downexpression group (p=0.008). Multivariate analysis showed that Tiam1 expression was a significant and independent prognostic parameter (p=0.042) for HCC patients. Tiam1 expression may be a novel and independent predictor for the prognosis of HCC patients.

[Effect of Tiam-l gene silencing on human colorectal carcinoma cell line SW480 metastasis in nude mice observed by real-time whole-body fluorescence imaging].

OBJECTIVE: To observe the effect of Tiam-l gene silencing on the metastasis of human colorectal carcinoma cell line SW480 in nude mice by real-time whole-body fluorescence imaging. METHODS: Enhanced green fluorescence protein (EGFP)-labeled human colorectal carcinoma cells, SW480/EGFP(+)/Tiam-1(-) and SW480/EGFP(+), were implanted into nude mice via tail vein injection or orthotopic colonal inoculation, and real-time whole-body fluorescence imaging was performed to compare the difference in tumor progression and metastasis between the two cells. RESULTS: Both SW480/EGFP(+) and SW480/ EGFP(+)/Tiam-1(-) cells stably expressed EGFP, and Tiam1 gene expression was reduced in SW480/EGFP(+)Tiam-1(-) to 30% of the expression level in SW480/EGFP(+) cells. The growth rate of the two cell lines had no significant difference in vitro (P>0.05), but SW480/EGFP(+)/Tiam1(-) cell proliferation and metastasis were depressed obviously in comparison with SW480/EGFP(+) in vivo (P<0.05). CONCLUSION: Tiam-1 gene may play an important role in invasion and metastasis of human colorectal cancer.