Comparison of myopic astigmatic correction after cross-assisted SMILE, FS-LASIK, and transPRK.

PURPOSE: To compare astigmatic correction among cross-assisted small-incision lenticule extraction (SMILE), femtosecond laser-assisted in situ keratomileusis (FS-LASIK), and transepithelial photorefractive keratectomy (transPRK). SETTING: The Eye Hospital of Wenzhou Medical University, Zhejiang, China. DESIGN: Prospective comparison study. METHODS: 154 right eyes of 154 patients with astigmatism of -1.00 to -2.75 diopters (D) were included in this study. 64 eyes, 42 eyes, and 48 eyes were receiving SMILE, FS-LASIK, and transPRK, respectively. The SMILE group used cross-axial alignment for head positioning for astigmatism correction. In the FS-LASIK and transPRK groups, static and dynamic cyclotorsion control were used. Changes in ocular parameters and vector analysis were assessed at 6 months postoperatively. RESULTS: The safety and efficacy indices were comparable among the 3 groups at 6 months postoperatively. Residual astigmatism was smallest in the SMILE group (-0.23 ± 0.25 D) compared with that in FS-LASIK (-0.40 ± 0.28 D, P = .009) and transPRK groups (-0.42 ± 0.32 D, P = .001). 53 (82.8%), 36 (85.7%), and 37 (77.1%) eyes achieved an angle of error within ±5 degrees, respectively ( P = .55). Notably, vector analysis showed that the difference vector, the magnitude of the error, and its absolute value were significantly smaller in the SMILE group than those in the other groups ( P < .05). In addition, the higher-order aberrations, especially coma, were significantly induced postoperatively in each group ( P < .001). CONCLUSIONS: Residual astigmatism magnitude was smallest by cross-assisted SMILE, followed by FS-LASIK and transPRK, and the astigmatism axial correction was comparable among groups.

KLF5 and p53 comprise an incoherent feed-forward loop directing cell-fate decisions following stress.

In response to stress, cells make a critical decision to arrest or undergo apoptosis, mediated in large part by the tumor suppressor p53. Yet the mechanisms of these cell fate decisions remain largely unknown, particularly in normal cells. Here, we define an incoherent feed-forward loop in non-transformed human squamous epithelial cells involving p53 and the zinc-finger transcription factor KLF5 that dictates responses to differing levels of cellular stress from UV irradiation or oxidative stress. In normal unstressed human squamous epithelial cells, KLF5 complexes with SIN3A and HDAC2 repress TP53, allowing cells to proliferate. With moderate stress, this complex is disrupted, and TP53 is induced; KLF5 then acts as a molecular switch for p53 function by transactivating AKT1 and AKT3, which direct cells toward survival. By contrast, severe stress results in KLF5 loss, such that AKT1 and AKT3 are not induced, and cells preferentially undergo apoptosis. Thus, in human squamous epithelial cells, KLF5 gates the response to UV or oxidative stress to determine the p53 output of growth arrest or apoptosis.

Comparison of Astigmatic Correction With and Without Cross-axis Alignment During Small Incision Lenticule Extraction.

PURPOSE: To compare clinical outcomes after astigmatism correction via small incision lenticule extraction (SMILE) with and without cross-axis alignment. METHODS: This prospective study included patients who underwent SMILE with astigmatism of greater than 0.75 diopters (D). In the alignment group, head position was readjusted by cross-axis alignment before the standard SMILE procedure. First, the cross-axis was aligned to corresponding green lines on the headrest. Then, the patient's head was adjusted to align the horizontal line to the outer canthus of both eyes and align the vertical line connecting the midpoints of the eyebrows and the bridge of the nose. Changes in ocular parameters were assessed, and vector analysis was performed 6 months postoperatively. RESULTS: The alignment and control groups included 61 and 54 eyes, respectively. Postoperatively, the safety and efficacy indices were comparable between the two groups. Notably, refractive cylinder differed significantly in the alignment group (-0.23 ± 0.26 D) compared to the control group (-0.36 ± 0.26 D) (P = .007). Forty-eight (78.7%) and 32 (59.3%) eyes in the alignment and control groups (P = .03) achieved an angle of error within ±5°, respectively. Vector analysis showed a significantly lower difference vector and a significantly better index of success in the alignment group than that in the control group (0.24 ± 0.25 vs 0.35 ± 0.24, P = .003 and 0.20 ± 0.22 vs 0.29 ± 0.22, P = .02, respectively). Moreover, the change in corneal trefoil differed significantly between the groups (P < .001). CONCLUSIONS: Cross-axis alignment for head positioning in SMILE significantly minimizes axis misalignment and reduces undercorrection astigmatism in myopic astigmatism correction. This technique is a non-invasive and effective method, especially for beginners. [J Refract Surg. 2022;38(10):624-631.].

Characteristics of the Optic Nerve Head in Myopic Eyes Using Swept-Source Optical Coherence Tomography.

Purpose: To investigate the characteristics of the optic nerve head (ONH) in myopia using swept-source optical coherence tomography (SS-OCT). Methods: Participants were divided into three groups according to the axial length (AL). The optic disc morphology, retinal nerve fiber layer (RNFL) thickness, and radial peripapillary capillary (RPC) vessel density (VD), optic disc tilt, rotation, Bruch's membrane opening distance (BMOD), border length (BL), border tissue angle, focal lamina cribrosa (LC) defects, ß- and γ-zone peripapillary atrophy (PPA), microvasculature dropout (MvD), choroidal thickness (CT), and the choroidal vascularity index (CVI) were compared. Linear regression analysis evaluated relationships between spherical equivalent, AL, and ONH parameters. Results: One hundred five, 98, and 118 eyes were included in groups 1, 2, and 3, respectively. With AL increasing, the mean, superior and temporal CT, central mean and temporal, pericentral mean, inferior and nasal RPC VD, and temporal CVI decreased, whereas the mean and temporal RNFL thickness, optic disc, RIM and ß-PPA area, presence and area of γ-PPA, BMOD and BL increased. Compared to other groups, group 3 depicted a larger cup area, more focal LC defect and total and juxtapapillary MvD; a lower central superior, inferior and nasal, pericentral superior, and temporal RPC VD. Group 1 demonstrated more tilted disc, larger inferior and nasal CT, mean, superior, inferior, and nasal CVI. Conclusions: Myopia eyes have larger ONH changes, PPAs, regional RNFL, and MvD, but smaller regional CTs, RPC VD, and CVIs. SS-OCT may be useful in detecting ONH variations during myopia.

Non-invasive monitoring of the kinetic infiltration and therapeutic efficacy of nanoparticle-labeled chimeric antigen receptor T cells in glioblastoma via 7.0-Tesla magnetic resonance imaging.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell therapy is a promising treatment strategy in solid tumors. In vivo cell tracking techniques can help us better understand the infiltration, persistence and therapeutic efficacy of CAR T cells. In this field, magnetic resonance imaging (MRI) can achieve high-resolution images of cells by using cellular imaging probes. MRI can also provide various biological information on solid tumors. METHODS: The authors adopted the amino alcohol derivatives of glucose-coated nanoparticles, ultra-small superparamagnetic particles of iron oxide (USPIOs), to label CAR T cells for non-invasive monitoring of kinetic infiltration and persistence in glioblastoma (GBM). The specific targeting CARs included anti-human epidermal growth factor receptor variant III and IL13 receptor subunit alpha 2 CARs. RESULTS: When using an appropriate concentration, USPIO labeling exerted no negative effects on the biological characteristics and killing efficiency of CAR T cells. Increasing hypointensity signals could be detected in GBM models by susceptibility-weighted imaging MRI ranging from 3 days to 14 days following the injection of USPIO-labeled CAR T cells. In addition, nanoparticles and CAR T cells were found on consecutive histopathological sections. Moreover, diffusion and perfusion MRI revealed significantly increased water diffusion and decreased vascular permeability on day 3 after treatment, which was simultaneously accompanied by a significant decrease in tumor cell proliferation and increase in intercellular tight junction on immunostaining sections. CONCLUSION: These results establish an effective imaging technique that can track CAR T cells in GBM models and validate their early therapeutic effects, which may guide the evaluation of CAR T-cell therapies in solid tumors.

Geochemistry and tectonic significance of late Paleoproterozoic A-type granites along the southern margin of the North China Craton.

The Longwangzhuang pluton is a typical example of Paleoproterozoic A-type granite intrusions at the southern margin of the North China Craton. This pluton is composed of arfvedsonite granite and minor aegirine-augite granites. Samples from both granite types display similar zircon U-Pb ages with 207U-206Pb ages of 1612 ± 19 Ma [mean square weighted deviation (MSWD) = 0.66] and 1609 ± 24 Ma (MSWD = 0.5), respectively. The granites exhibit similar high silica (SiO2 = 71.1-73.4 wt.%), high alkaline (Na2O + K2O = 8.10-9.26 wt.%, K2O/Na2O > 1), and low Al2O3 (11.8-12.8 wt. %) contents and metaluminous to weakly peraluminous bulk chemistry. The chemical variations of the Longwangzhuang pluton suggest the effects of mineral fractionation. In addition, all samples show typical characteristics of A-type granites, such as high 10000Ga/Al ratios (4.10-7.28), high FeOtot/(FeOtot + MgO) ratios (0.88-0.99), and high Zr (484-1082 ppm), Ce (201-560 ppm), and Y (78-156 ppm) contents. The εNd(t) values and the (206Pb/204Pb)t, (207Pb/204Pb)t, and (208Pb/204Pb)t ratios of the arfvedsonite granite samples vary from -4.6 to -5.3, 15.021 to 17.349, 15.241 to 15.472, and 33.206 to 36.905, respectively, and those for the aegirine-augite granite sample amount at -0.2, 14.421, 15.175, and 33.706. The distinct and variable Nd and Pb isotope values indicate the presence of heterogeneous protoliths. Based on its geochemistry, its low initial Pb isotope ratios, and its enrichment in Nd isotopes, we infer that the Longwangzhuang A-type granite is the partial melting product of basement rocks such as the Taihua Group gneisses accompanied by some involvement of juvenile material from the mantle. Together with published data from other Paleoproterozoic A-type granite plutons exposed at the southern margin of the craton, our findings suggest that these granites had a similar origin. Furthermore, geochemically, they can be divided into two groups: A2-type, which formed earlier (~1.8-1.6 Ga), and A1-type, which formed later (~1.6-1.5 Ga). Combining this information with the variations in whole-rock Nd and zircon Hf isotopic composition at ca. 1.6 Ga, we propose that tectonic transformation from post-orogenic to anorogenic magmatism occurred at the southern margin of the North China Craton at that time.

Microvascular characteristics of lower-grade diffuse gliomas: investigating vessel size imaging for differentiating grades and subtypes.

OBJECTIVES: Vessel size imaging (VSI) could reveal average microvessel diameter. The aim was to investigate microvascular characteristics and the efficacy of VSI in lower-grade glioma (LGG) grading and subtype differentiation based on 2016 classification of central nervous system tumours. METHODS: Fifty-seven LGG (grade II/III, 36/21) patients who received VSI examination before surgery were retrospectively analysed. The average (Rmean) and maximum (Rmax) vessel size indexes were obtained. The long (VDmax) and short (VDmin) vascular diameter, microvascular area (MVA) and density (MVD) were obtained using paraffin specimens. The patients were divided into grades II and III, and histological and molecular subtypes. The differences among microvascular parameters of different subtypes and grades were compared. Two-sample t-test, analysis of variance test, Mann-Whitney test, the Kruskal-Wallis test and Pearson correlation analysis were used for statistics. RESULTS: Rmean, Rmax, VDmin, VDmax, and MVA were higher in grade-III than in grade-II LGGs (p < 0.05) in each type except the isocitrate dehydrogenase (IDH) mutant with 1p/19q-intact type. For grade II, the IDH mutant with 1p/19q co-deleted and IDH wildtype possessed more dominant angiogenesis than IDH mutant with 1p/19q-intact type, revealed by lower Rmean, Rmax and VDmin while higher MVD for the former (p < 0.05), the same as oligodendroglioma versus astrocytoma. Rmean and Rmax correlated with VDmin (r = 0.804, 0.815, p < 0.05), VDmax (r = 0.766, 0.774, p < 0.05) and MVA (r = 0.755, 0.759, p < 0.05), respectively, while they had no correlation with MVD (r = -0.085, -0.080, p > 0.05). CONCLUSIONS: VSI holds great potential for non-invasively revealing microvascular characteristics of LGGs pre-surgery and differentiating their grades and molecular subtypes. KEY POINTS: • VSI can assist in differentiating grade-II and -III gliomas. • The IDH gene and 1p/19q chromosome may influence the angiogenesis in grade-II gliomas. • VSI is valuable for differentiating the molecular subtypes of grade-II gliomas.

Dexamethasone pretreatment impairs the thymidylate synthase inhibition mediated flare in thymidine salvage pathway activity in non-small cell lung cancer.

INTRODUCTION: Successful inhibition of thymidylate synthase (TS) by pemetrexed, a TS inhibitor, results in a reproducible transient burst or "flare" in thymidine salvage pathway activity at 2 hrs. of therapy which can be measurable with FLT-PET ([18F]fluorothymidine-positron emission tomography) in non-small cell lung cancer (NSCLC). Routine administration of dexamethasone with pemetrexed-based therapy could potentially confound this imaging approach since dexamethasone is known to inhibit expression of thymidine kinase 1, a key enzyme in the thymidine salvage pathway. Here we examine the potential impact of dexamethasone on the TS inhibition-mediated thymidine salvage pathway "flare" in NSCLC. MATERIALS AND METHODS: In order to determine NSCLC cell line sensitivity to dexamethasone and pemetrexed, IC50 studies were performed on NSCLC cell lines H23, H1975, H460, H1299. TS inhibition-mediated "flare" in thymidine salvage pathway activity was then measured at 2hrs. of exposure to pemetrexed and cisplatin in NSCLC cells lines following using 3H-thymidine incorporation assays under the following conditions: control (no chemotherapy or dexamethasone), or treated with pemetrexed and cisplatin without dexamethasone, with 24 hrs. pre-treatment of dexamethasone or with dexamethasone administered together with chemotherapy. These conditions were chosen to model the delivery of pemetrexed-based therapy in the clinic. RESULTS: The IC50 of H23, H1975, H460, H1299 for dexamethasone and pemetrexed were 40, 5.9, 718, 362 µM and 0.22, 0.73, 0.14 and 0.66 µM respectively. Significant blunting of the thymidine salvage pathway "flare" is observed at 2hrs. of pemetrexed-based therapy when dexamethasone sensitive cell lines H23 and H1975 were pretreated with dexamethasone but not when dexamethasone was given together with pemetrexed therapy or in the setting of dexamethasone resistance (H460 and H1299). CONCLUSION: 24 hr. pretreatment with dexamethasone, but not same day co-administration of dexamethasone with therapy, impairs the TS inhibition-mediated "flare" in thymidine salvage pathway activity in NSCLC.

Dynamic MR imaging for functional vascularization depends on tissue factor signaling in glioblastoma.

Glomeruloid vascular proliferation (GVP) is a diagnostic hallmark and links to aggressive behavior, therapy resistance and poor prognosis in glioblastoma (GBM). It lacks clinical approaches to predict and monitor its formation and dynamic change. Yet the mechanism of GVPs also remains largely unknown. Using an in situ GBM xenograft mouse model, combined clinical MRI images of pre-surgery tumor and pathological investigation, we demonstrated that the inhibition of tissue factor (TF) decreased GVPs in Mouse GBM xenograft model. TF shRNA reduced microvascular area and diameter, other than bevacizumab. TF dominantly functions via PAR2/HB-EGF-dependent activation under hypoxia in endothelial cells (ECs), resulting in a reduction of GVPs and cancer cells invasion. TF expression strongly correlated to GVPs and microvascular area (MVA) in GBM specimens from 56 patients, which could be quantitatively evaluated in an advanced MRI images system in 33 GBM patients. This study presented an approach to assess GVPs that could be served as a MRI imaging biomarker in GBM and uncovered a molecular mechanism of GVPs.

Textural features of dynamic contrast-enhanced MRI derived model-free and model-based parameter maps in glioma grading.

BACKGROUND: Presurgical glioma grading by dynamic contrast-enhanced MRI (DCE-MRI) has unresolved issues. PURPOSE: The aim of this study was to investigate the ability of textural features derived from pharmacokinetic model-based or model-free parameter maps of DCE-MRI in discriminating between different grades of gliomas, and their correlation with pathological index. STUDY TYPE: Retrospective. SUBJECTS: Forty-two adults with brain gliomas. FIELD STRENGTH/SEQUENCE: 3.0T, including conventional anatomic sequences and DCE-MRI sequences (variable flip angle T1-weighted imaging and three-dimensional gradient echo volumetric imaging). ASSESSMENT: Regions of interest on the cross-sectional images with maximal tumor lesion. Five commonly used textural features, including Energy, Entropy, Inertia, Correlation, and Inverse Difference Moment (IDM), were generated. RESULTS: All textural features of model-free parameters (initial area under curve [IAUC], maximal signal intensity [Max SI], maximal up-slope [Max Slope]) could effectively differentiate between grade II (n = 15), grade III (n = 13), and grade IV (n = 14) gliomas (P < 0.05). Two textural features, Entropy and IDM, of four DCE-MRI parameters, including Max SI, Max Slope (model-free parameters), vp (Extended Tofts), and vp (Patlak) could differentiate grade III and IV gliomas (P < 0.01) in four measurements. Both Entropy and IDM of Patlak-based Ktrans and vp could differentiate grade II (n = 15) from III (n = 13) gliomas (P < 0.01) in four measurements. No textural features of any DCE-MRI parameter maps could discriminate between subtypes of grade II and III gliomas (P < 0.05). Both Entropy and IDM of Extended Tofts- and Patlak-based vp showed highest area under curve in discriminating between grade III and IV gliomas. However, intraclass correlation coefficient (ICC) of these features revealed relatively lower inter-observer agreement. No significant correlation was found between microvascular density and textural features, compared with a moderate correlation found between cellular proliferation index and those features. DATA CONCLUSION: Textural features of DCE-MRI parameter maps displayed a good ability in glioma grading. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:1099-1111.

Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma.

Notch1 transactivates Notch3 to drive terminal differentiation in stratified squamous epithelia. Notch1 and other Notch receptor paralogs cooperate to act as a tumor suppressor in squamous cell carcinomas (SCCs). However, Notch1 can be stochastically activated to promote carcinogenesis in murine models of SCC. Activated form of Notch1 promotes xenograft tumor growth when expressed ectopically. Here, we demonstrate that Notch1 activation and epithelial-mesenchymal transition (EMT) are coupled to promote SCC tumor initiation in concert with transforming growth factor (TGF)-ß present in the tumor microenvironment. We find that TGFß activates the transcription factor ZEB1 to repress Notch3, thereby limiting terminal differentiation. Concurrently, TGFß drives Notch1-mediated EMT to generate tumor initiating cells characterized by high CD44 expression. Moreover, Notch1 is activated in a small subset of SCC cells at the invasive tumor front and predicts for poor prognosis of esophageal SCC, shedding light upon the tumor promoting oncogenic aspect of Notch1 in SCC.

Clinical Applications of Contrast-Enhanced Perfusion MRI Techniques in Gliomas: Recent Advances and Current Challenges.

Gliomas possess complex and heterogeneous vasculatures with abnormal hemodynamics. Despite considerable advances in diagnostic and therapeutic techniques for improving tumor management and patient care in recent years, the prognosis of malignant gliomas remains dismal. Perfusion-weighted magnetic resonance imaging techniques that could noninvasively provide superior information on vascular functionality have attracted much attention for evaluating brain tumors. However, nonconsensus imaging protocols and postprocessing analysis among different institutions impede their integration into standard-of-care imaging in clinic. And there have been very few studies providing a comprehensive evidence-based and systematic summary. This review first outlines the status of glioma theranostics and tumor-associated vascular pathology and then presents an overview of the principles of dynamic contrast-enhanced MRI (DCE-MRI) and dynamic susceptibility contrast-MRI (DSC-MRI), with emphasis on their recent clinical applications in gliomas including tumor grading, identification of molecular characteristics, differentiation of glioma from other brain tumors, treatment response assessment, and predicting prognosis. Current challenges and future perspectives are also highlighted.

Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging.

INTRODUCTION: Inhibition of thymidylate synthase (TS) results in a transient compensatory "flare" in thymidine salvage pathway activity measureable with 18F-thymidine (FLT)- positron emission tomography (PET) at 2hrs. of therapy which may predict non-small cell lung cancer (NSCLC) sensitivity to TS inhibition. MATERIALS AND METHODS: Resistance to TS inhibition by pemetrexed was induced in NSCLC cell lines H460 and H1299 through TS overexpression. TS overexpression was confirmed with RT-PCR and Western blotting and pemetrexed resistance confirmed with IC50 assays. The presence of a pemetrexed-induced thymidine salvage pathway "flare" was then measured using 3H-thymidine in both pemetrexed sensitive (H460 and H1299) and resistant (H460R, H1299R, CALU-6, H522, H650, H661, H820, H1838) lines in vitro, and validated with FLT-PET in vivo using H460 and H460R xenografts. RESULTS: Overexpression of TS induced pemetrexed resistance with IC50 for H460, H1299, H460R and H1299R measured as 0.141 µM, 0.656 µM, 22.842 µM, 213.120 µM, respectively. Thymidine salvage pathway 3H-thymidine "flare" was observed following pemetrexed in H460 and H1299 but not H460R, H1299R, CALU-6, H522, H650, H661, H820 or H1838 in vitro. Similarly, a FLT "flare" was observed in vivo following pemetrexed therapy in H460 but not H460R tumor-bearing xenografts. CONCLUSIONS: Imaging of TS inhibition is predictive of NSCLC sensitivity to pemetrexed.

Quantitative in vivo imaging of tissue factor expression in glioma using dynamic contrast-enhanced MRI derived parameters.

OBJECTIVE: Tissue Factor (TF) has been well established in angiogenesis, invasion, metastasis, and prognosis in glioma. A noninvasive assessment of TF expression status in glioma is therefore of obvious clinical relevance. Dynamic contrast-enhanced (DCE) MRI parameters have been used to evaluate microvascular characteristics and predict molecular expression status in tumors. Our aim is to investigate whether quantitative DCE-MRI parameters could assess TF expression in glioma. MATERIALS AND METHODS: Thirty-two patients with histopathologically diagnosed supratentorial glioma who underwent DCE-MRI were retrospectively recruited. Extended Tofts linear model was used for DCE-MRI post-processing. Hot-spot, whole tumor cross-sectional approaches, and histogram were used for analysis of model based parameters. Four serial paraffin sections of each case were stained with TF, CD105, CD34 and α-Sooth Muscle Actin, respectively for evaluating the association of TF and microvascular properties. Pearson correlation was performed between percentage of TF expression area and DCE-MRI parameters, multiple microvascular indexes. RESULTS: Volume transfer constant (Ktrans) hot-spot value best correlated with TF (r=0.886, p<0.001), followed by 90th percentile Ktrans value (r=0.801, p<0.001). Moreover, histogram analysis of Ktrans value demonstrated that weak TF expression was associated with less heterogeneous and positively skewed distribution. Finally, pathology analysis revealed TF was associated with glioma grade and significantly correlated with these two dynamic angiogenic indexes which could be used to explain the strong correlation between Ktrans and TF expression. CONCLUSION: Our results indicate that Ktrans may serve as a potential clinical imaging biomarker to predict TF expression status preoperatively in gliomas.

Early detection of pemetrexed-induced inhibition of thymidylate synthase in non-small cell lung cancer with FLT-PET imaging.

Inhibition of thymidylate synthase (TS) results in a transient "flare" in DNA thymidine salvage pathway activity measurable with FLT ([18F]thymidine)-positron emission tomography (PET). Here we characterize this imaging strategy for potential clinical translation in non-small cell lung cancer (NSCLC). Since pemetrexed acts by inhibiting TS, we defined the kinetics of increases in thymidine salvage pathway mediated by TS inhibition following treatment with pemetrexed in vitro. Next, using a mouse model of NSCLC, we validated the kinetics of the pemetrexed-mediated "flare" in thymidine salvage pathway activity in vivo using FLT-PET imaging. Finally, we translated our findings into a proof-of-principle clinical trial of FLT-PET in a human NSCLC patient. In NSCLC cells in vitro, we identified a burst in pemetrexed-mediated thymidine salvage pathway activity, assessed by 3H-thymidine assays, thymidine kinase 1 (TK1) expression, and equilibrative nucleoside transporter 1 (ENT1) mobilization to the cell membrane, that peaked at 2hrs. This 2hr time-point was also optimal for FLT-PET imaging of pemetrexed-mediated TS inhibition in murine xenograft tumors and was demonstrated to be feasible in a NSCLC patient. FLT-PET imaging of pemetrexed-induced TS inhibition is optimal at 2hrs from therapy start; this timing is feasible in human clinical trials.

KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification.

Epithelial differentiation and stratification are essential for normal homeostasis, and disruption of these processes leads to both injury and cancer. The zinc-finger transciption factor KLF4 is a key driver of epithelial differentiation, yet the mechanisms and targets by which KLF4 controls differentiation are not well understood. Here, we define WNT5A, a non-canonical Wnt ligand implicated in epithelial differentiation, repair, and cancer, as a direct transcriptional target that is activated by KLF4 in squamous epithelial cells. Further, we demonstrate functionally that WNT5A mediates KLF4 control of epithelial differentiation and stratification, as treatment of keratinocytes with WNT5A rescues defective epithelial stratification resulting from KLF4 loss. Finally, we show that the small GTPase CDC42 is regulated by KLF4 in a WNT5A dependent manner. As such, we delineate a novel pathway for epithelial differentiation and stratification and define potential therapeutic targets for epithelial diseases.

KLF4 is downregulated but not mutated during human esophageal squamous cell carcinogenesis and has tumor stage-specific functions.

The transcriptional regulator Krüppel-like factor 4 (KLF4) is decreased in human esophageal squamous cell cancer (ESCC), and Klf4 deletion in mice produces squamous cell dysplasia. Nonetheless the mechanisms of KLF4 downregulation in ESCC and the functions of KLF4 during ESCC development and progression are not well understood. Here, we sought to define the regulation of KLF4 and delineate the stage-specific effects of KLF4 in ESCC. We found that KLF4 expression was decreased in human ESCC and in 8 of 9 human ESCC cell lines. However, by genomic sequencing, we observed no KLF4 mutations or copy number changes in any of 52 human ESCC, suggesting other mechanisms for KLF4 silencing. In fact, KLF4 expression in human ESCC cell lines was increased by the DNA methylation inhibitor 5-azacytidine, suggesting an epigenetic mechanism for KLF4 silencing. Surprisingly, while KLF4 decreased in high-grade dysplasia and early stage tumors, KLF4 increased with advanced cancer stage, and KLF4 expression in ESCC was inversely correlated with survival. Interestingly, KLF4 promoted invasion of human ESCC cells, providing a functional link to the stage-specific expression of KLF4. Taken together, these findings suggest that KLF4 loss is necessary for esophageal tumorigenesis but that restored KLF4 expression in ESCC promotes tumor spread. Thus, the use of KLF4 as a diagnostic and therapeutic target in cancer requires careful consideration of context.

The expression of P2X7 receptors in EPCs and their potential role in the targeting of EPCs to brain gliomas.

In order to use endothelial progenitor cells (EPCs) as a therapeutic and imaging probe to overcome antiangiogenic resistance for gliomas, how to enhance proliferation and targeting ability of transplanted EPCs is a high priority. Here, we confirmed, for the first time, the expression of P2X7 receptors in rat spleen-derived EPCs. Activation of P2X7 receptors in EPCs by BzATP promoted cells proliferation and migration, rather than apoptosis. In vivo, the homing of transplanted EPCs after long-term suppression of P2X7 receptors by persistent BBG stimulation was evaluated by MRI, immunohistochemistry and flow cytometry. Compared to the group without BBG treatment, less transplanted EPCs homed to gliomas in the group with BBG treatment, especially integrated into the vessels containing tumor-derived endothelial cells in gliomas. Moreover, western blot showed that CXCL1 expression was downregulated in gliomas with BBG treatment, which meant P2X7 receptors suppression inhibited the homing of EPCs to gliomas through down-regulation of CXCLl expression. Further, effects of P2X7 receptors on C6 glioma cells or gliomas were evaluated at the same dose of BzATP or BBG used in EPCs experiments in vitro and in vivo. MTT assay and MRI revealed that P2X7 receptors exerted no significant promoting effect on C6 glioma cells proliferation, gliomas growth and angiogenesis. Taken together, our findings imply the possibility of promoting proliferation and targeting ability of transplanted EPCs to brain gliomas in vivo through P2X7 receptors, which may provide new perspectives on application of EPCs as a therapeutic and imaging probe to overcome antiangiogenic resistance for gliomas.

A new mosaic pattern in glioma vascularization: exogenous endothelial progenitor cells integrating into the vessels containing tumor-derived endothelial cells.

Emerging evidence suggests that glioma stem-like cells (GSCs) transdifferentiating into vascular endothelial cells (ECs) possibly contributes to tumor resistance to antiangiogenic therapy. Endothelial progenitor cells (EPCs), showing active migration and incorporation into neovasculature of glioma, may be a good vehicle for delivering genes to target GSCs transdifferentiation. Here, we found a new mosaic pattern that exogenous EPCs integrated into the vessels containing the tumor-derived ECs in C6 glioma rat model. Further, we evaluated the effect of these homing EPCs on C6 glioma cells transdifferentiation. The transdifferentiation frequency of C6 glioma cells and the expressions of key factors on GSCs transdifferentiation, i.e. HIF-1α, Notch1, and Flk1 in gliomas with or without EPCs transplantation showed no significant difference. Additionally, magnetic resonance imaging could track the migration and incorporation of EPCs into glioma in vivo, which was confirmed by Prussian blue staining. The number of magnetically labeled EPCs estimated from T2 maps correlated well with direct measurements of labeled cell counts by flow cytometry. Taken together, our findings may provide a rational base for the future application of EPCs as a therapeutic and imaging probe to overcome antiangiogenic resistance for glioma and monitor the efficacy of this treatment.

Krüppel-like factors in cancer.

Krüppel-like factors (KLFs) are a family of DNA-binding transcriptional regulators with diverse and essential functions in a multitude of cellular processes, including proliferation, differentiation, migration, inflammation and pluripotency. In this Review, we discuss the roles and regulation of the 17 known KLFs in various cancer-relevant processes. Importantly, the functions of KLFs are context dependent, with some KLFs having different roles in normal cells and cancer, during cancer development and progression and in different cancer types. We also identify key questions for the field that are likely to lead to important new translational research and discoveries in cancer biology.