Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Side and top views of the culture dish before setup. The dish was a normal 12-phi glass-bottom dish. A glass separator was set at the center of the dish with a bioinert adhesive. (b) First, we placed 150 μl fibrin gel mixed with HUVECs in the center of the glass plate, so that two sides of the dish were separated by the glass separator and fibrin gel. We also added LF-containing fibrin gel at the edge of the dish. (c) We incubated the dish for 30 min to solidify the fibrin gel. (d) We added 1 ml culture medium to both wells and incubated the dish for 1 week. (e) After 1 week of culture, a vascular network with a perfusable lumen was formed in the glass-bottom region. Then, we cut both edges of the regions to make openings. (f) After the cuts, we increased the amount of culture medium on one side of the dish. This caused a static pressure difference between both openings of the self-organized capillary network, resulting in steady flow inside the apparatus. (g) Low magnification view of the self-organized vascular network. RFP-HUVECs were cultivated in the fibrin gel, and we observed the vascular network formation. (h) High magnification view of (g). Vascular network with a lumen was generated in the fibrin gel region. (i) Low magnification view of the self-organized vascular network after long-term culture with flow. (j) Visualization of the perfusable area by FITC-dextran. Perfusable regions existed near the inlet and outlet, near the glass separator and edge of the well. (k) Flow inside the lumen was visualized using fluorescent beads. (l) Snapshot of the culture system when using whole blood as a tracer. Red blood cells were flowing inside the self-organized vasculature. (m) Projection of multiple frames of (l). Movements of red blood cells were visualized as a stream. Scale bars: 3 mm (g, i, j); 100 μm (h); 500 μm (k); 50 μm (l, m).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques: Adhesive, Incubation, Generated

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Experimental procedure. Spheroids containing RFP-HUVECs and lung fibroblast were generated and embedded in fibrin gel. After 1 week, sprouts from the spheroids became sufficiently long. Then, we cut the tip of the sprouts from both sides of the well and exerted static pressure to one side of the well. (b) Visualization of the perfusion inside the spheroid using FITC-dextran. (c) High-magnification view of the sprouts connecting two spheroids. FITC-dextran is running through the sprout structure. Scale bar: 1 mm (b); 100 μm (c).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques: Generated

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Initial shape of the vascular network. HUVECs were stained with UEA1 and nuclei were stained with Hoechst 33342. There was a flow-positive region (yellow-dashed line) and non-flow region (green-dashed line). Direction of flow is indicated by a white allow. (b) Kymograph of the yellow-dashed line region. Horizontal direction represents space, and vertical direction represents time. Collective movement toward upstream of the flow was observed. White arrow indicates the flow of cell debris. (c) Kymograph of the green-dashed line region. Cell movement was random. (d–n) Cell shape changes induced by flow: (d, e) Fast flow. When FITC dextran was perfused, the vessel regions near the inlet or outlet showed fast flow. (f) Brightfield view of the fast flow region. Endothelial cells became shaped as spindles aligned parallel to the flow direction. (g) Fluorescence view of the fast flow region. At the floor of the lumen, we observed spindle-shaped cells parallel to the flow direction. (h, i) Slow flow. When FITC dextran was perfused, the vessel regions far from the inlet or outlet showed slow flow. (j) Brightfield view and (k) confocal view of the slow flow region. Endothelial cells did not show any polarity. (l) Low magnification view of the non-flow region. (m) Brightfield view and (n) confocal view of the non-flow region. Vasculatures were disconnected and became thin endothelial cysts with cell debris inside. Scale bars: 1 mm (d, e, h, i, l); 200 μm (f, g, j, k, m, n).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques: Staining, Fluorescence

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Experiment setup. We prepared two groups of dishes in which a mixture of RFP-HUVECs and NMuMG-Fucci cells were seeded in the fibrin gel. After the perfusable network was formed, we made openings to both groups but transferred medium in only one group of the dishes. (b) Typical appearance of the NMuMG cell colony and its schematic representation. NMuMG-Fucci cells (red and green nuclei) forms colonies outside the vascular lumen. (c) Time course of the cell division monitored by Fucci reporter. (d) Time course of GFP(+) area ratio. 20 NMuMG-Fucci colonies were observed, and the area of GFP(+) areas was obtained using Fiji. (e) GFP(+) area ratio between day 1 and day 0. Statistically significant difference was detected (Student t-test). Scale bars: 50 μm.

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques:

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) LM4-GFP cells were introduced into the self-organized capillary network consisting of HUVECs visualized by UEA-1 lectin. (b) High magnification time-lapse view of (a). We observed cancer cells emigrating out of the blood vessels. (c) Detailed morphology of cancer cells on the endothelial cells. Emigrated LM4 cells attached to the blood vessel with highly polarized morphology and multiple protrusions. (c) Max projection image, (c') orthogonal section, and (c'') 3D-reconstructed image. White arrows: cancer cell protrusions. Scale bars: 50 μm (a); 10 μm (b, c).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques:

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 1. The SM22α-Cre specificity in the mouse brain vasculature. mT/mG reporter mice were bred with SM22α-Cre deleter mice (mT/mG:SM22α-Cre), and brain tissues were harvested at postnatal day 6 (P6). A, Sections of cerebellum and cerebrum were detected for mG expression by fluorescence microscopy. SM22α-Cre-driven mG was specifically detected in microvessels of both cerebrum and cerebellum (arrowheads) but not in control mT/mG mice (arrows). High power images of arrowhead-indicated regions are shown on the right. n=3 mice per group. B, Cerebral sections were immunostained with anti- PDGFR-β followed by an allophycocyanin (APC)-conjugated secondary antibody with an IgG isotype as a control. mG expression was colocalized with the pericyte (PC) marker PDGFR-β in the brain microvasculature of mT/mG:SM22α-Cre (arrowhead), but not in the IgG staining or in the control mT/mG mice (arrow). n=3 mice per group. C, mG+ and mG− cell populations were isolated from P6 mT/mG:SM22α- Cre brain tissues, and gene expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with specific PC and endothelial cell (EC) markers as indicated. mG+ cells expressed PC marker genes PDGFRB (PDGFR-β, SCPG4 [NG-2], and ANPEP [CD13], but not EC marker genes PECAM1 [CD31], VEGFR2 [VEGFR2] and CDH5 [VE-cadherin]) with normalization by GAPDH. Data are mean±SEM; n=3; ***P<0.001 by unpaired 2-tailed Student t test. D and E, Mouse brain microvascular pericytes (mBMVPCs) and ECs (mBMVECs) were isolated from wild-type (WT) mice at P6, and immunostained with PC marker PDGFR-β and EC marker VE-cadherin. Phase images (D) and immunofluorescence images (E) are presented. F–I, Ccm3 deletion was specifically in mouse brain PCs but not in mouse brain ECs. mBMVPCs and mBMVECs were isolated from P6 WT and Ccm3smKO brain tissues. F, Cells were immunostained with PC marker PDGFR-β and EC marker VE-cadherin. G, Ccm3 gene expression was determined by qRT-PCR. n=3; ***P<0.001 by unpaired 2-tailed Student t test. H, CCM3 protein was determined by Western blotting. Representative blot form 3 experiments. I, CCM3 protein was determined by immunostaining using an anti-CCM3 antibody with costaining of antipaxillin antibody. n=3. Scale bar: 50 μm (A, B, and D); 25 μm (E and F); 10 μm (I).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Expressing, Fluorescence, Microscopy, Control, Marker, Staining, Isolation, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Western Blot, Immunostaining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 3 Continued. Representative images are shown in E. Quantification of % GFAP coverage on CD31+-vessel was quantified by Image J (F). G–I, Mouse brain microvascular pericytes (mBMVPCs) were isolated from neonatal WT and CCM3 smKO brains. 4×105 WT and CCM3- knockout (KO) mBMVPCs were seeded on fibronectin-coated culture slides for indicated times (0–16 h), and unattached cells were washed away. Cells were fixed with 4% paraformaldehyde (PFA) and stained with phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Representative images for each time point are shown (G). Cell area (H) and cell length (I) were measured by Image J software. Ten fields were counted and n=3 repeated experiments. J and K, RNA-seq analyses. The endogenous CCM3 was knocked out by CRISPR/Cas9. mRNA from confluent WT and CCM3-KO human brain microvascular pericytes (hBMVPCs) were subjected to RNA-seq analyses. J, Gene expression value was estimated by Cufflinks (v1.2.0) and genes with >2-fold change between WT and KO were defined as differential expression. K, Gene Ontology analysis using GOstats was performed and the significant pathways (muscle cell migration and extracellular matrix [ECM] organization) are presented. n=2. Data are means±SEM. Scale bars: 25 μm (A, C, G, and I); 500 nm (E).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Isolation, Knock-Out, Staining, Software, RNA Sequencing, CRISPR, Gene Expression, Quantitative Proteomics, Migration

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 5. Cerebral cavernous malformation (CCM)3-knockout (KO) pericytes (PCs) attenuates PC migration and endothelial cell (EC)-PC interactions. CCM3-KO human brain microvascular pericytes (hBMVPCs) were re-expressed with vector (VC), CCM3-wild type (WT), or CCM3-4KE by lentivirus infection. A, WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were harvested and subjected to Western blotting to test for adhesion complexes and RhoA-pMLC signaling. Relative protein levels were quantified and fold changes are presented by keeping WT as 1.0. B and C, 4×105 hBMVPCs were seeded on fibronectin-coated culture slides for 16 h. Cells were fixed with 4% paraformaldehyde (PFA) followed costaining with phosphor-paxillin (green) and phalloidin (red) with DAPI counterstaining (blue) (B). Number of FA per cell was measured by Image J software. Ten fields were counted and n=3 repeated experiments. D and E, Rescue PC migration by CCM3-WT but not by paxillin-defective CCM3-4KE mutant. WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were subjected to wound injury followed by incubation for 24 h. D, Representative images of cell migration are shown. Dashed lines indicate the remaining gaps. E, Quantitation of EC migration. The percentage of unhealed wound was quantified, n=3. F and G, EC-PC interactions in 3-dimensional spheroid sprouting assay. Human brain microvascular ECs (hBMVECs) were infected with EGFP (enhanced green fluorescent protein)-expressing retroviruses, whereas WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were infected with mCherry-expressing lentiviruses. ECs and PCs (2:1 ratio) were seeded to beads and coated with microbeads, embedded in fibrin gels and grown in EGM-2 endothelial growth medium for 4 d. A representative image of 10 beads for each sample is shown in F and percentage of PC coverage of sprouts is quantified in G. n=10, *P<0.05; **P<0.01 (1- way ANOVA). Additional 2 independent experiments were performed. Error bars indicate SEM. Scale bar: 10 μm (B); 100 μm (D and F).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Knock-Out, Migration, Plasmid Preparation, Infection, Western Blot, Software, Mutagenesis, Incubation, Quantitation Assay, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 6. Cerebral cavernous malformation (CCM)3 loss in pericyte (PC) induces extracellular matrix (ECM) deposition in CCM. A and B, Increased ECM deposition in CCM3-deficient human brain microvascular pericytes (hBMVPCs). Wild-type (WT) and CCM3-KO hBMVPCs were cultured confluently on fibronectin-coated culture slides for 16 h. Cells were subjected to costaining with fibronectin (green) and Col IV (red) with DAPI counterstaining (blue). Mean fluorescence intensity (MFI)/per cell were measured by Image J software. Ten fields were counted and n=3 repeated experiments. C and D, P6 WT and Ccm3smKO mouse brain sections for staining of CD31 with fibronectin or NG-2. IgG isotype was used as a control. Representative images are shown (C). Normalized fibronectin MFI were measure by Image J (by taking WT as 1.0). Scale bar: 50 μm (A and C). E, A model for CCM3-depleted PC in promoting CCM lesion progression. The brain microvessels have an extraordinarily high PC to endothelial cell (EC) ratio, and PCs have multiple slender processes extending longitudinally to cover capillary EC for vascular integrity. Integrin-mediated matrix interactions and PC migration are critical in this process. Focal adhesion- mediated cell adhesion is important for cell migration, but the extent of adhesion can govern migration speed. We observed that CCM3- deficient PCs exhibit excess adhesion due to enhanced ECM deposition, ITG-β1 (integrin β1) activation and paxillin-mediated focal adhesion. We propose that CCM3 loss in PCs enhances PC adhesion while reducing PC protrusion and migration along EC, leading to the disruption of PC-EC interactions and resulting in CCM lesion formation.

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Cell Culture, Fluorescence, Software, Staining, Control, Migration, Activation Assay, Disruption

Journal: Advanced healthcare materials

Article Title: Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

doi: 10.1002/adhm.202400388

Figure Lengend Snippet: Figure 5. Effects of vasculogenesis inhibitors on interactions between HRMVECs and HRMVPs. a) 3D image analysis routine to quantify cell-cell contacts (Imaris, Oxford Instruments). 1) The relevant channels are filtered (Gaussian), 2) a channel-specific surface algorithm is performed, 3) the Imaris Xtension ‘surface surface contact area’ is applied for creating a surface at the junction of both cell types. 4) Close-up of contacts (yellow) between HRMVECs (CellTracker Orange, green) and HRMVPs (GFP, orange) (scale bar 1–3. 200 μm and 4. 50 μm). Total filament lengths for b) HRMVECs and c) cocultured HRMVPs. d) Percentage of all HRMVECs surfaces in contact with HRMVPs. e) Total number of contacts. f) Total surface area of contacts. Data presented as violin plots with horizontal lines indicating quartiles 1–3 (n = 4–6). Asterisks indicate multiplicity adjusted P values of one-way ANOVA with post hoc Tukey test, comparing inhibitor treatments to respective vehicle controls; *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Human telomerase reverse transcriptase (hTert) immortalized human retinal microvascular cells (HRMVECs) and hTert immortalized GFPexpressing human retinal microvascular pericytes (HRMVPs), as well as non-fluorescent hTert immortalized HRMVPs (Angio-Proteomie, US), were cultured on tissue culture flasks pre-coated with quick coating solution (Angio-Proteomie, US) in endothelial growth medium and pericyte growth medium (Angio-Proteomie, US) at 37 °C and 5% CO2 in a humidified incubator, respectively.

Techniques:

Journal: Alzheimer's & Dementia

Article Title: Structural changes in cerebral microvasculature induced by ferroptosis contribute to blood–brain barrier disruption in Alzheimer's disease: an autopsy study

doi: 10.1002/alz.70103

Figure Lengend Snippet: Associations between ABC score and microvascular pathology.

Article Snippet: In this experiment, human microvascular pericytes (HVPCs, Catalog No.: HUM‐iCell‐n011) were obtained from iCell ( http://www.icellbioscience.com ) and cultured using a specialized primary human brain microvascular pericyte medium (Catalog No.: iCell‐n011‐002 h); the hCMEC/D3 cell line (Catalog No.: CL‐0843) was purchased from Pricella ( https://www.procell.com.cn ) and cultured using a specialized hCMEC/D3 medium (Catalog No.: CM‐0843).

Techniques:

Journal: Alzheimer's & Dementia

Article Title: Structural changes in cerebral microvasculature induced by ferroptosis contribute to blood–brain barrier disruption in Alzheimer's disease: an autopsy study

doi: 10.1002/alz.70103

Figure Lengend Snippet: The BBB of AD brains is damaged with elevated agrin deposition and reduced pericytes. (A) Immunohistochemical staining shows that the deposition of agrin is severer (bar = 300 µm) while PDGFRβ is lower (bar = 50 µm) in the AD group. The inverted mean gray values of the area immunoreactive for DAB staining were measured from five images (1 × 1 mm) captured randomly using ImageJ software. The data are presented as median and interquartile range. Statistical analysis conducted using Mann–Whitney U test. Antibody used: agrin (Sigma‐Aldrich, MAB5204, 1:500), PDGFRβ (Proteintech, 13449‐1‐AP, 1:500). (B) Immunofluorescence shows that claudin‐5 (green) and agrin (red) are colocalized in pericytes (yellow), and blue indicates DAPI (bar = 10 µm). Antibody used: claudin‐5 (Proteintech, 29767‐1‐AP, 1:500), agrin (Sigma‐Aldrich, MAB5204, 1:500). (C) Immunofluorescence shows that agrin (green) exposure led to a reduction in PDGFRβ (red) levels (bar = 50 µm). (D) Fluorescence semiquantitative analysis of agrin and PDGFRβ. We randomly selected three 450 × 450 µm visual fields for statistical analysis ( n = 6) using two‐way ANOVA, followed by Bonferroni's multiple comparisons test. The data are presented as the means ± SEM. (E) Semiquantitative analysis of PDGFRβ and claudin‐5 protein levels. Statistical analysis conducted using two‐way ANOVA, followed by Bonferroni's multiple comparisons test. Data are presented as means ± SEM. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: In this experiment, human microvascular pericytes (HVPCs, Catalog No.: HUM‐iCell‐n011) were obtained from iCell ( http://www.icellbioscience.com ) and cultured using a specialized primary human brain microvascular pericyte medium (Catalog No.: iCell‐n011‐002 h); the hCMEC/D3 cell line (Catalog No.: CL‐0843) was purchased from Pricella ( https://www.procell.com.cn ) and cultured using a specialized hCMEC/D3 medium (Catalog No.: CM‐0843).

Techniques: Immunohistochemical staining, Staining, Software, MANN-WHITNEY, Immunofluorescence, Fluorescence

Journal: Alzheimer's & Dementia

Article Title: Structural changes in cerebral microvasculature induced by ferroptosis contribute to blood–brain barrier disruption in Alzheimer's disease: an autopsy study

doi: 10.1002/alz.70103

Figure Lengend Snippet: Agrin treatment of pericytes led to increased generation of ROS and ferroptosis. (A) CCK‐8 assay was used to detect proliferation of pericytes after stimulation with different concentrations of agrin (5, 10, or 50 µg/mL) for various durations (1, 2, 6, 12, and 24 h) ( n = 3). Statistical analysis conducted using two‐way ANOVA. (B) Fluorescence microscopy observation and detection of fluorescent probe for ROS, which is used to show the changes in ROS expression in pericytes caused by agrin stimulation (bar = 100 µm). The data are presented as the means ± SEM ( n = 4) and statistical analysis conducted using Mann–Whitney U test. (C) Flow cytometry to detect effect of agrin on pericyte apoptosis by initially staining cells with annexin V and propidium iodide (PI) solution. Cells that were PI negative and annexin V‐positive cells are considered apoptotic. Data are presented as mean ± SEM ( n = 4), and statistical analysis conducted using two‐way ANOVA. (D) Representative fluorescence images of intracellular Fe 2 ⁺ levels in four groups are shown (bar = 50 µm). Data presented as mean ± SEM ( n = 4). Statistical analysis was performed using one‐way ANOVA with post hoc testing. (E) Semiquantitative analysis of GPX4 and FTH1 protein levels. Data are presented as mean ± SEM. Statistical analysis was conducted using two‐way ANOVA, followed by Bonferroni's multiple comparisons test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: In this experiment, human microvascular pericytes (HVPCs, Catalog No.: HUM‐iCell‐n011) were obtained from iCell ( http://www.icellbioscience.com ) and cultured using a specialized primary human brain microvascular pericyte medium (Catalog No.: iCell‐n011‐002 h); the hCMEC/D3 cell line (Catalog No.: CL‐0843) was purchased from Pricella ( https://www.procell.com.cn ) and cultured using a specialized hCMEC/D3 medium (Catalog No.: CM‐0843).

Techniques: CCK-8 Assay, Fluorescence, Microscopy, Expressing, MANN-WHITNEY, Flow Cytometry, Staining

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: Model creation and colorectal cancer-secreted cytokine profiling ( A) Schematic of hydrogel model creation and cluster analysis of CRC cell line cytokine profiles. Media containing secreted cytokines and exosomes of Caco2, SW480, and HCT116 CRC cell lines was isolated via a centrifugation protocol. Normal human fibroblasts or microvascular pericytes were suspended in collagen I-hyaluronic acid hydrogel matrices and cultured in CRC conditioned media to study cellular response to primary tumor soluble signals. (B–C) Clustering analysis of conditioned media based on a 200 cytokine array showed distinct signatures for conditioned media produced by each cell line. Cytokines upregulated in metastatic cell-conditioned media and their expression levels are shown in (C). CM – Caco2 Media, SM – SW480 Media, HM – HCT116 Media, FM – Fibroblast Control Media, PM – Pericyte Control Media.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Isolation, Centrifugation, Cell Culture, Produced, Expressing, Control

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: CRC Secretome Differentially Activates Pericytes and Fibroblasts in 3D Hydrogel Constructs. CRC-derived factors differentially activate fibroblasts and pericytes in the hydrogel microenvironment (A) Representative images of phalloidin (green)/α-SMA (red)/DAPI (blue) stain on fibroblasts (top) and pericytes (bottom) treated for 3 days with colorectal cancer conditioned media. (B and C) Eccentricity quantification of fibroblasts and pericytes in each condition, where a score of 0 indicates circularity, and a score nearing one indicates a spindle-like shape. (D and E) Fluorescence quantification of α-SMA expression per cell. Scale bar 50 μm. Significance: ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Construct, Derivative Assay, Staining, Fluorescence, Expressing

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: Pericytes Exhibit a Relaxed Phenotype in Response to CRC-Secreted Factors CRC-derived factors promote pericyte relaxation and changes in cell-matrix interactions. (an i-iv) Representative images of pericytes stained for phosphorylated myosin light chain (green, iii.), paxillin (orange, iv.), and cell nuclei (blue, ii.) after three days of culture in conditioned media. (B) Fluorescence quantification of p -MLC. Phosphorylation of myosin light chain decreases significantly in pericytes exposed to HCT116 CM as compared to control conditions. (C) Fluorescence quantification of paxillin. No significant change in paxillin expression was observed for any condition, despite visual differences in staining patterns. Scale bar 20 μm. Significance: ∗∗∗p < 0.001. Data are represented as mean ± SEM

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Derivative Assay, Staining, Fluorescence, Phospho-proteomics, Control, Expressing

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: CRC Secretome Drives Matrix Metalloproteinase Signaling and Collagen Deposition CCSF exposure changes collagen degradation and deposition behaviors for both pericytes and fibroblasts. (A–C) SW480 conditioned media significantly increases MMPs two and nine expressions by fibroblasts. All CRC media upregulate MMP13 expression, with HCT116 media inducing the largest change. (D–F) Pericyte MMP2 expression is significantly downregulated with exposure to CCSFs, and additionally correlates with CRC cell line aggression. Pericyte MMP13 is underexpressed with CCSF exposure, and MMP9 shows no clear trend relative to media type. (G and H) HCT116 conditioned media significantly increases the quantity of collagen deposition by fibroblasts; pericytes follow the same trend but have no significant changes. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM n ≥ 3.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Expressing

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet: Pericytes and Fibroblasts Reorganize Collagen Fiber Architecture and Density in the Presence of CCSFs Picrosirius red stain of collagen fibers in fibroblast and pericyte-laden hydrogels.(A) Polarized light images of picrosirius red stains were analyzed using CT-Fire software. (B) Collagen fiber density was slightly increased for all CCSF conditions in fibroblast cultures, and significantly lowered for SW480 and HCT116 conditions in pericyte cultures. (C) Collagen fiber width skewed lower in fibroblasts CCSF cultures compared to normal control and higher in pericyte SW480 and HCT116 cultures. Fiber length was not affected by CCSF exposure in either fibroblast or pericyte cultures. Collagen alignment was lower, i.e., was less organized, in fibroblast and pericyte hydrogels cultured with HCT116 media. Scale bar 50 μm. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Bar plots are shown as mean ± SEM Box and whisker plots are presented with Tukey formatting and horizontal bar at the median.

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Staining, Software, Control, Cell Culture, Whisker Assay

Journal: iScience

Article Title: Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model

doi: 10.1016/j.isci.2022.104645

Figure Lengend Snippet:

Article Snippet: Normal human lung fibroblasts (NHLF CC-2512, Lonza, Morristown, NJ) and human microvascular pericytes (#1200, ScienCell, San Diego, CA) were expanded in tissue-culture treated plastic dishes.

Techniques: Recombinant, Collagen Assay, Staining, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics, Software