cell growth kit vegf  (ATCC)

Journal: Bioactive Materials

Article Title: Mesenchymal stromal cells-loaded 3D radially aligned composite scaffold with potentiated paracrine signaling for sequential bone regeneration

doi: 10.1016/j.bioactmat.2026.02.059

Figure Lengend Snippet: Temporal analysis of the BMSC paracrine profile on different scaffolds. (A) Confocal microscopy images from Live/Dead fluorescence staining of BMSCs encapsulated within the PCL/HAp-GelMA/BMSCs scaffold after 1, 3, 5, and 14 d of 3D culture (live cells, green; dead cells, red). (B) The concentrations of key paracrine factors (TGF-β, PGE2, VEGF, HGF, and BMP-2) from BMSCs cultured in different scaffolds, quantified from culture supernatants at day 3 and day 7. (C) Corresponding relative mRNA expression levels of TGFB1, PTGS2, VEGFA, HGF, and BMP-2 in BMSCs at day 3 and day 7, as determined by qPCR analysis. Data are presented as mean ± SD (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns: not significant.

Article Snippet: ELISA kits for PGE2 (Cat. No. E-EL-0034), TGF-β (Cat. No. E-EL-0162), VEGF (Cat. No. E-EL-R2603), and HGF (Cat. No. E-EL-R0496) were purchased from Elabscience (Wuhan, China).

Techniques: Confocal Microscopy, Fluorescence, Staining, Cell Culture, Expressing

Journal: bioRxiv

Article Title: CXCL-CXCR2 signaling drives cancer-endothelium interactions in SCLC metastatic seeding

doi: 10.64898/2026.04.15.716394

Figure Lengend Snippet: a. Schematic illustrating isolation of monocultured and SCLC co-cultured liver sinusoidal endothelial cells (LSECs) for single-cell RNA sequencing (scRNA-seq). Primary human LSECs labeled with BFP were co-cultured for 24 hours with mCherry-labeled H82 SCLC cells at a 2:1 ratio. CD31□ BFP□ mCherry□ LSECs were isolated by flow cytometry and subjected to scRNA-seq. b. scRNA-seq identifies distinct transcriptional states in SCLC co-cultured LSECs. Left : UMAP visualization of five LSEC clusters (EC1-EC5) derived from monoculture and two independent H82 co-culture replicates. A total of 41,278 high-quality cells were retained for analysis, with an average sequencing depth of 19,427 UMIs per cell. Right : Condition-split UMAPs show that LSECs exposed to SCLC undergo marked transcriptional reprogramming relative to monoculture. Notably, the cluster 4 LSEC population (EC4) emerges specifically in co-culture conditions. c. SCLC co-culture expands EC2 and EC4 LSEC populations. Alluvial plots depict shifts in LSEC cluster composition between monoculture and co-culture conditions. Both H82 co-culture replicates show a remarkable increase in EC2 and EC4 populations, accompanied by a corresponding decrease in EC1 and EC3 populations. d. RNA velocity analysis identifies EC1 as the progenitor population that gives rise to EC2 and EC4. UMAP embeddings of LSEC scRNA-seq data with RNA velocity streamlines overlaid demonstrate directional trajectories originating from EC1 and progressing toward EC2 and EC4 under co-culture conditions. e. LSEC clusters exhibit distinct transcriptional programs. Heat map of LSEC gene expression showing the top five marker genes per cluster, generated using a downsampled set of 2,000 cells per cluster. Color denotes normalized expression levels. Each endothelial cluster displays a unique marker gene signature, delineating five discrete transcriptional states within the LSEC compartment. f. SCLC co-cultured LSECs show strong CXCL expression. Feature plots map CXCL chemokine expression onto UMAP embeddings of monocultured and co-cultured LSECs. Notably, CXCL expression is markedly enriched in both co-culture replicates and absent in the monoculture condition. g. Cluster EC4 shows activation of inflammation related pathways. Heat map showing cluster average normalized enrichment score of MSigDB Hallmark gene sets averaged across five EC clusters. Notably, EC4 displays specific enrichment of inflammation-related pathways. h. CXCL1 and CXCL2 are upregulated in HUVECs co-cultured with H82 cells. Volcano plot showing differentially expressed genes (DEGs) in HUVECs cultured alone versus co-cultured with H82 SCLC cells. DEGs with an adjusted p-value less than 1×10□¹□ and absolute log□ fold change more than 1 are highlighted in red. CXCL1 and CXCL2 are among the most strongly upregulated genes in the co-culture condition. i. Upregulation of CXCL1, CXCL2, and CXCL3 is conserved across endothelial cell types. Scatter plot comparing log□ fold changes for differentially expressed genes in monocultured versus H82 co-cultured LSECs (scRNA-seq) and HUVECs (bulk RNA-seq). CXCL1, CXCL2, and CXCL3 are consistently upregulated in both endothelial populations under co-culture conditions, highlighting a conserved SCLC-induced inflammatory chemokine response. j. Liver endothelial cells upregulate CXCL1 in SCLC-transplanted mice. Left : Representative immunofluorescence images of CD31□ endothelial cells in the brain, lung, and liver of sham-treated or RP48 SCLC–transplanted mice. CXCL1 signal is most strongly increased in liver endothelial cells 48 hours after RP48 SCLC transplantation. Right : Quantification of CXCL1 immunofluorescence across tissues, shown as the mean ± SD ratio of CXCL1 to DAPI signal intensity per field (N = 20 fields). k. SCLC induces CXCL1 protein expression in co-cultured HUVECs. Left : Representative immunofluorescence images of HUVECs maintained in monoculture or co-cultured with H82 SCLC cells show an increase in CXCL1 protein signal in the co-culture condition. Right : Quantification of CXCL1 expression per field, presented as the mean ± SD ratio of CXCL1 to DAPI signal intensity per field (N = 10 fields). l. Representative spatial transcriptomic map showing liver LSECs and spatial co-expression of CXCL2 with the LSEC marker CLEC4M in the liver parenchyma of colorectal cancer (CRC) liver metastasis. m. CXCL chemokines are predominantly produced by liver LSECs in metastatic lesions. Bar plots comparing CXCL2, CXCL5, and CXCL6 expression between LSECs and alveolar ECs from CRC liver and lung metastases show significantly higher expression of all three CXCL transcripts in LSECs. n. Heatmap of three CXCL chemokines included in the Xenium 5K panel (CXCL2, CXCL5, CXCL6) demonstrating higher expression in liver ECs compared with lung ECs across eight liver metastasis and six lung metastasis samples.

Article Snippet: HUVECs were cultured in Vascular Cell Basal Medium (ATCC, PCS-100-030) with Endothelial Cell Growth Kit (ATCC, PCS-100-041); All cell lines were confirmed to be mycoplasma negative (MycoAlert Detection Kit, Lonza).

Techniques: Isolation, Cell Culture, Single Cell, RNA Sequencing, Labeling, Flow Cytometry, Derivative Assay, Co-Culture Assay, Sequencing, Gene Expression, Marker, Generated, Expressing, Activation Assay, Immunofluorescence, Transplantation Assay, Produced

Journal: bioRxiv

Article Title: CXCL-CXCR2 signaling drives cancer-endothelium interactions in SCLC metastatic seeding

doi: 10.64898/2026.04.15.716394

Figure Lengend Snippet: a. CXCL chemokines do not affect SCLC cell viability. Viability of H82 and RP48 SCLC cells treated with recombinant human or mouse CXCL1, CXCL2, or CXCL3 was quantified using a CCK-8 assay. Data are presented as mean ± SD of OD□□□ fold change relative to vehicle-treated controls (N = 4 wells). b. CXCL chemokines enhance migration of non-adherent SCLC cells. Recombinant CXCL1, CXCL2, or CXCL3 was placed in the lower chamber, and non-adherent H82 ( Left ) or RP48 ( Right ) SCLC cells were seeded in the upper chamber. Migrated cells were quantified from 10% of the lower chamber and presented as mean ± SD (N = 3 wells). c. CXCL chemokines enhance migration of adherent SCLC cells. Left: Representative images showing migration of adherent H446 and DMS-273 SCLC cells toward vehicle control or CXCL chemokines in the lower chamber. Right: Migrated cells per well are quantified and reported as mean ± SD (N = 6 wells). d. Schematic of SCLC migration toward EC-conditioned media (EC-CM). EC-CM generated from HUVEC monoculture or HUVEC-H82 co-culture was placed in the lower transwell chamber, and mCherry-labeled H82 cells in the upper chamber were assessed for migration following 48 hours of incubation. e. Co-culture-derived EC-CM promotes SCLC migration. EC-CM collected from HUVEC-H82 co-cultures yielded a higher number of migrated SCLC cells relative to HUVEC monoculture-derived EC-CM. Migrated cells were quantified from 10% of the lower chamber and presented as mean ± SD (N = 4 wells). f. Co-culture-derived EC-CM promotes transwell migration of adherent SCLC cell lines. Left: Representative images of transwell migration of H446 and DMS-273 SCLC cells toward EC-CM derived from HUVEC monoculture and HUVEC-H82 co-culture. Right: Migrated cells per well are quantified and reported as mean ± SD (N = 6 wells). g. Schematic of HEK-293-SCLC co-culture transwell migration assay. GFP□ HEK-293 cells overexpressing CXCL1, CXCL2, or CXCL3 were placed in the lower chamber of a transwell system, and mCherry□ H82 SCLC cells were seeded in the upper chamber. Migration was quantified by flow cytometry as the percentage of mCherry□ H82 cells detected in the lower chamber relative to GFP□ HEK-293 cells. h. CXCL-expressing HEK-293 cells enhance SCLC migration independent of endothelial cell identity. Flow cytometry analysis ( Left ) and quantitative assessment ( Right ) show increased migration of H82 cells toward HEK-293 cells overexpressing CXCL1, CXCL2, or CXCL3, compared with control HEK-293 cells. Migration is normalized to the number of GFP□ HEK-293 cells in the lower chamber. Ratios of mCherry□ H82 to GFP□ HEK-293 cells per well are reported as mean ± SD (N = 4 wells). i. Schematic of a 3D microfluidic SCLC-EC co-culture system mimicking trans-endothelial migration. The blood channel (BC) is lined with HUVECs and contains mCherry-labeled SCLC cells, while the side channel (SC) is filled with medium supplemented with CXCL chemokines. The two channels are separated by a hydrogel (HG) matrix. SCLC trans-endothelial migration is assessed by quantification of SCLC cells that traverse the HUVEC-lined barrier and enter the hydrogel toward CXCLs in the side channel. The entire device is maintained under orbital shaking to generate physiologically relevant shear stress. j. CXCL factors stimulate H82 trans-endothelial migration. Left : Representative images showing mCherry-labeled H82 cells migrating into the hydrogel in the absence or presence of CXCL chemokines. Right : Quantification of H82 trans-endothelial migration. The presence of CXCL factors is essential for SCLC extravasation into the hydrogel. Data reported as mean ± SD of mCherry + H82 per chip (N = 3 chips). k. CXCLs induce F-actin assembly in SCLC cells. Left : Representative immunofluorescence images of F-actin staining in RP48 and DMS-273 SCLC cell lines treated with or without CXCL chemokines. Right : Quantification of F-actin signal per cell, presented as mean ± SD of the F-actin to DAPI signal intensity ratio per field (N > 15 fields). CXCL treatment significantly increased F-actin assembly in both SCLC cell lines. l. Schematic of quantification of SCLC-EC interactions via the G-baToN system. H82 SCLC cells (mCherry□) expressing cell surface GFP served as sender cells, while HUVECs (BFP□) expressing cell surface anti-GFP served as receiver cells. GFP transfer during direct cell-cell contact was assessed by flow cytometry, and cultures were subjected to orbital shaking to model physiological shear stress. m. CXCL factors strengthen interactions between H82 SCLC cells and HUVECs. Left: Flow cytometry analysis of GFP transfer in monocultured HUVEC receivers and in co-cultures of H82 senders with HUVEC receivers, with or without CXCLs supplementation. Right : Quantification of SCLC-EC interaction strength, measured as the percentage of GFP□ cells within the viable BFP□mCherry□ HUVEC receiver population (mean ± SD; N = 3). CXCL treatment significantly increased GFP transfer into HUVECs, indicating enhanced physical contact between H82 cells and HUVECs. n. Schematic of quantification of CXCL-dependent effects on SCLC-HEK-293 interactions. Cell surface mCherry-expressing H82 SCLC cells (senders) and anti-mCherry-expressing HEK-293 cells (receivers) were co-cultured with or without additional overexpression of CXCL chemokines, under orbital shaking to mimic physiological shear stress. Direct H82-HEK-293 contact enabled mCherry transfer to HEK-293 receiver cells, which was quantified by flow cytometry as a measure of interaction strength. o. CXCL overexpression strengthens interactions between H82 cells and HEK-293 cells. Left : Flow cytometry analysis of mCherry transfer in monocultured HEK-293 receiver cells and in co-cultures of H82 sender cells with HEK-293 receivers expressing GFP control, CXCL1, or CXCL2. Right : Quantification of H82-HEK-293 interaction strength, measured as the percentage of mCherry□ cells within the viable GFP□ HEK-293 receiver population (mean ± SD; N = 3). CXCL overexpression significantly increased HEK-293 interaction with SCLC independently of endothelial identity.

Article Snippet: HUVECs were cultured in Vascular Cell Basal Medium (ATCC, PCS-100-030) with Endothelial Cell Growth Kit (ATCC, PCS-100-041); All cell lines were confirmed to be mycoplasma negative (MycoAlert Detection Kit, Lonza).

Techniques: Recombinant, CCK-8 Assay, Migration, Control, Generated, Co-Culture Assay, Labeling, Incubation, Derivative Assay, Transwell Migration Assay, Flow Cytometry, Expressing, Shear, Immunofluorescence, Staining, Cell Culture, Over Expression

Journal: bioRxiv

Article Title: CXCL-CXCR2 signaling drives cancer-endothelium interactions in SCLC metastatic seeding

doi: 10.64898/2026.04.15.716394

Figure Lengend Snippet: a. Schematic of quantitative assessment of CXCR2 function in SCLC metastasis using Metastasis-Originated Barcode Sequencing (MOBA-seq). Cas9-expressing RP48 SCLC cells were randomly barcoded with a lentiviral sgRNA library containing one control sgRNA and three sgRNAs targeting Cxcr2 . Barcoded SCLC cells were subsequently transplanted intravenously into recipient mice, and metastatic tissues were harvested at multiple time points. Genomic DNA from metastatic lesions in target organs was subjected to barcode sequencing and analyzed using the MOBA-seq pipeline to quantify genotype-specific contributions to metastatic seeding and outgrowth. b. Cxcr2 inactivation suppresses SCLC metastasis across multiple tissues and time points. Metastatic colonies recovered from mouse brain, liver, and lung at four time points were plotted, with each dot representing an individual barcode-derived metastatic colony and dot size scaled to colony cell number. Across all organs and time points, Cxcr2 inactivation reduced the number of metastatic colonies compared with controls. Pre-transplantation barcode distributions showed similar cell numbers between the two genotypes. c. Cxcr2 inactivation suppresses SCLC metastatic burden across multiple tissues and time points. Metastatic burden in brain, liver, and lung was quantified via MOBA-seq at 2 days, 1 week, 2 weeks, and 3 weeks post-transplantation. For each tissue and time point, relative metastatic burden was plotted as fold change compared with the control sgRNA. Data points outlined in black denote comparisons with statistical significance (FDR < 0.05). Cxcr2 inactivation reduced metastatic burden in multiple organs, with the most pronounced suppression observed in the liver. d. Cxcr2 inactivation suppresses SCLC metastatic seeding across multiple tissues and time points. Relative metastatic seeding was quantified in brain, liver, and lung at 2 days, 1 week, 2 weeks, and 3 weeks post-transplantation. Fold changes in metastatic seeding were plotted relative to sg Ctrl . Data points outlined in black denote statistical significance (FDR < 0.05). Cxcr2 inactivation led to a marked reduction in the number of seeded metastatic colonies across all tissues and time points. e. MOBA-seq identifies dormant and expanding metastatic colonies across tissues. The distribution of log□-transformed metastatic colony sizes from liver, lung, and brain at the 3-week time point was modeled to identify two key features: a dormant peak mode (left mode, red) and a valley mode (right mode, blue) demarcating the transition from dormant to expanding colonies. These empirically derived cutoff values were uniformly applied across all three tissues to enable unbiased quantification of genotype-specific effects on metastatic dormancy. Notably, RP48 cells showed evidence of escaping dormancy only in the liver. f. Cxcr2 inactivation increases metastatic dormancy in the liver, but not lung, at 3 weeks. A Gaussian mixture model was applied to the log□-transformed metastatic colony size distributions from liver and lung, using the dormant-mode and valley-mode cutoffs defined in ( e ) to quantify genotype-specific dormancy in each organ. In the liver , Cxcr2 inactivation substantially increased the proportion of dormant colonies compared with control. In contrast, Cxcr2 inactivation led to a reduction in dormant colonies in the lung. g. Cxcr2 inactivation suppresses dissemination of metastatic SCLC cells into the blood. Shared barcodes detected in both liver metastases and circulating tumor cells were used to identify metastatic colonies capable of further dissemination (“SuperMets,” highlighted in red). Left : Jitter plots of colony sizes across time points show the frequency and distribution of SuperMet colonies for each genotype. Right : Size distributions of SuperMet versus regular metastatic colonies. Cxcr2 inactivation reduced the number of SuperMet colonies and increased the colony size threshold for dissemination. h. Schematic showing MOBA-seq comparison of the metastatic effects of CXCR2 and established metastatic drivers in human SCLC. Barcoded H82-Cas9 SCLC cells were transduced with a lentiviral sgRNA library containing a control sgRNA and sgRNAs targeting CXCR2 , CXCR4 , or NFIB . After intravenous transplantation of the barcoded pools into recipient mice, metastasis-bearing liver tissues were harvested for library preparation, sequencing, and analysis. MOBA-seq was performed to quantify genotype-specific metastatic fitness and evaluate the relative effect of CXCR2 compared with known metastatic drivers CXCR4 and NFIB . i. CXCR2 inactivation suppresses liver metastasis at 3 weeks, comparable to inactivation of other metastatic driver genes. Metastatic colonies recovered from liver at 3 weeks were plotted, with each dot representing a single barcode-derived colony and dot size scaled to colony cell number. Inactivation of CXCR2 , CXCR4 , or NFIB each resulted in a reduction in metastatic colony number relative to control. Pre-transplantation barcode distributions confirmed similar input cell numbers across all genotypes. j. CXCR2 inactivation suppresses metastatic burden as strongly as NFIB . Relative metastatic burden in the liver at 3 weeks was plotted as fold change compared with control sgRNA, with statistically significant differences (FDR < 0.05) shown in color. Both CXCR2 and NFIB inactivation resulted in significantly reduced metastatic burden. k. CXCR2 inactivation suppresses metastatic seeding more strongly than other metastatic driver genes. Relative metastatic seeding in the liver at 3 weeks was plotted as fold change compared with control sgRNA, with statistically significant differences (FDR < 0.05) shown in color. CXCR2 inactivation produced the greatest reduction in metastatic seeding, exceeding the effects observed with CXCR4 or NFIB inactivation. l. CXCR2 -dependent metastatic phenotypes are conserved between human and mouse SCLC cell lines. Radar plots summarizing liver metastatic metrics for Cxcr2 demonstrate similar suppression of metastatic burden, metastatic seeding, and dormancy escape in human H82 and mouse RP48 SCLC cell lines. Notably, inactivation of Cxcr2 in RP48 cells results in higher 90th-percentile colony sizes and peak mode values compared with H82 cells, indicating cell line-specific CXCR2 effects on clonal expansion dynamics. m. Schematic of quantification of CXCL chemokines as pro-metastatic factors in SCLC liver metastasis via MOBA-seq. Mice were treated by hydrodynamic injection (HDI) to deliver plasmids expressing either GFP alone (control) or GFP-tagged CXCL chemokines. 24 hours after HDI, barcoded SCLC cells were transplanted intravenously and metastases-bearing livers were harvested two weeks later. MOBA-seq was then performed to quantify the impact of CXCL overexpression on liver metastatic outgrowth. n. Hydrodynamic injection suppresses baseline SCLC liver metastasis. Left : Representative images of metastases-bearing mouse livers with and without HDI treatment show a marked reduction in H82 SCLC metastatic lesions in HDI-treated livers. Right : Quantification of metastasis-bearing liver weight per mouse demonstrates a significant decrease in metastatic burden following HDI treatment, presented as mean ± SD (N = 3 mice). o. Hydrodynamic injection efficiently transfects liver ECs. Flow cytometry analysis of GFP expression in liver CD31 + EC following HDI with either saline or CMV-GFP plasmid. HDI achieved robust transfection, with 35.9% of liver endothelial cells expressing GFP. p. CXCL overexpression restores SCLC metastatic seeding in HDI-treated mouse livers. Metastatic colonies recovered from HDI-treated mouse livers were plotted, with each dot representing an individual barcode-derived colony and dot size scaled to colony cell number. Overexpression of CXCL1, CXCL2, or CXCL3 increased the number of metastatic colonies relative to GFP control. q. CXCL1 and CXCL2 overexpression promote dormancy escape of SCLC metastasis. The log□□-transformed metastatic colony size distributions for GFP control and CXCL-overexpressing conditions were plotted. CXCL1- and CXCL2-overexpressing livers showed a shift toward expanding metastatic colonies compared with the predominantly dormant colony distribution observed in GFP control livers.

Article Snippet: HUVECs were cultured in Vascular Cell Basal Medium (ATCC, PCS-100-030) with Endothelial Cell Growth Kit (ATCC, PCS-100-041); All cell lines were confirmed to be mycoplasma negative (MycoAlert Detection Kit, Lonza).

Techniques: Sequencing, Expressing, Control, Derivative Assay, Transplantation Assay, Transformation Assay, Comparison, Transduction, Produced, Injection, Over Expression, Flow Cytometry, Saline, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: CXCL-CXCR2 signaling drives cancer-endothelium interactions in SCLC metastatic seeding

doi: 10.64898/2026.04.15.716394

Figure Lengend Snippet: a. Loss of CXCR2 does not affect SCLC cell viability. Viability of wild-type (WT) and CXCR2 -knockout (KO) H82 and RP48 SCLC cells was quantified using CCK-8 assay. After 72 hours of culture, no significant (n.s.) differences in cell viability were detected between WT and KO cells in either line. Data are presented as mean ± SD of OD□□□ fold change normalized to WT controls (N = 4 wells). b. Loss of CXCR2 suppresses CXCL-induced SCLC cell migration. Transwell migration assays were performed by adding vehicle or CXCL1, CXCL2, or CXCL3 to the lower chamber and seeding WT or KO H82 or RP48 SCLC cells in the upper chamber. Migrated SCLC cells were quantified from 20% of the lower chamber and are presented as mean ± SD (N = 3 wells). Loss of CXCR2 significantly reduced migration under both baseline (vehicle) conditions and all CXCL-stimulated conditions. c. Schematic of color-coded SCLC transwell competition assay. Equal numbers of GFP□ WT and mCherry□ CXCR2 -KO SCLC cells were seeded into the upper chamber of a transwell system, with CXCL chemokines added to the lower chamber. Following migration, the proportion of GFP□ WT and mCherry□ KO cells in the lower chamber was quantified by flow cytometry to assess competitive migratory advantage between the two populations. d. Loss of CXCR2 impairs competitive SCLC transwell migration. GFP-labeled WT and mCherry-labeled CXCR2-KO H82 or RP48 SCLC cells were mixed at a 1:1 ratio and subjected to the transwell competition assay. Data are presented as mean ± SD of the relative percentage of GFP□ and mCherry□ cells normalized to total cells (N = 3 wells). WT cells consistently outcompeted KO cells in migration toward CXCL-containing lower chambers. e. CXCR2 overexpression (OvE) enhances SCLC response to CXCL-induced migration. Transwell migration of GFP-labeled control and mCherry-labeled CXCR2-OvE H82 SCLC cells was quantified by flow cytometry under vehicle or CXCL-stimulated conditions. Data are presented as mean ± SD of the relative percentage of GFP□ and mCherry□ cells normalized to total cells (N = 3 wells). CXCR2 overexpression did not alter baseline (vehicle) migration but significantly increased CXCL-driven migration. f. CXCR2 overexpression enhances adherent SCLC response to CXCL-induced migration. Left : Representative transwell images of control and CXCR2-OvE adherent SCLC cell lines (DMS-273 and H446) under vehicle or CXCL-stimulated conditions. Right : Quantification of migrated SCLC cells per field for each condition, presented as mean ± SD (N = 5 fields). CXCR2 overexpression did not alter baseline migration but significantly increased migration in response to CXCL treatment. g. CXCR2 inactivation suppresses CXCL-induced F-actin assembly in SCLC cells. Left : Representative immunofluorescence images of WT and CXCR2-KO RP48 SCLC cells treated with vehicle or CXCLs, showing F-actin (red) and DAPI-stained nuclei (blue). Right : Quantification of F-actin levels per cell, presented as mean ± SD of the F-actin/DAPI signal intensity ratio per field (N > 15 fields). CXCL stimulation increased F-actin assembly in WT cells but not in CXCR2-KO cells. h. CXCR2 overexpression increases F-actin assembly in SCLC cells treated with CXCLs. Left : Representative immunofluorescence images of control and CXCR2-OvE RP48 SCLC cells showing F-actin (red) and DAPI-stained nuclei (blue). Right : Quantification of F-actin levels per cell, presented as mean ± SD of the F-actin/DAPI signal intensity ratio per field (N > 15 fields). CXCR2-OvE cells exhibited significantly enhanced F-actin organization compared with control cells. i. CXCR2 inactivation in SCLC cells decreases SCLC-endothelial cell interaction strength. Left : Flow cytometry analysis of GFP transfer in monocultured HUVEC receiver cells and in co-cultures with WT or CXCR2-KO H82 SCLC sender cells. Right : Quantification of the percentage of GFP□BFP□mCherry - HUVEC receiver cells when co-cultured with WT or CXCR2-KO H82 senders. Loss of CXCR2 significantly decreased GFP transfer into HUVEC receiver cells. j. Schematic of SCLC spontaneous liver metastasis via subcutaneous transplantation. Equal numbers of GFP-labeled CXCR2-WT cells and mCherry-labeled CXCR2-KO cells were injected into opposite flanks of mice to generate primary subcutaneous tumors. After four weeks, both subcutaneous primary tumors and metastasis-bearing liver tissues were collected, and the relative metastatic contribution of each genotype was quantified by comparing the abundance of GFP□ (WT) and mCherry□ (KO) cells. k. CXCR2 inactivation in SCLC cells does not impair primary subcutaneous tumor growth. Subcutaneous tumor volumes generated by WT and CXCR2-KO RP48 SCLC cells were quantified and are presented as mean ± SD (N = 5 mice). Primary tumor growth was comparable between WT and CXCR2-KO tumors. l. CXCR2 inactivation suppresses SCLC liver metastatic colonization. Representative fluorescence images of liver surface metastases (white arrows) derived from disseminated GFP□ WT and mCherry□ CXCR2 -KO RP48 SCLC cells. CXCR2 -KO cells exhibited markedly reduced metastatic colonization of the liver. m. CXCR2 inactivation reduces metastatic SCLC cell dissemination in the liver. Left : Flow cytometry analysis of GFP□ WT and mCherry□ CXCR2-KO RP48 SCLC cells isolated from metastasis-bearing livers four weeks after subcutaneous transplantation. Right : Quantification of the GFP□ to mCherry□ RP48 cell ratio per mouse liver, presented as mean ± SD (N = 5 mice). WT RP48 cells represented a significantly larger proportion of liver-disseminated SCLC cells compared with CXCR2-KO cells. n. CXCR2 inactivation prolongs survival and reduces liver metastatic colonization. Left : Kaplan-Meier survival curves of mice injected via tail vein with WT or CXCR2-KO RP48 SCLC cells. Loss of CXCR2 significantly extended overall survival relative to WT controls. Right : Representative necropsy images from mice that succumbed on day 43 illustrate the markedly reduced liver metastatic colonization in CXCR2-KO-recipient mice.

Article Snippet: HUVECs were cultured in Vascular Cell Basal Medium (ATCC, PCS-100-030) with Endothelial Cell Growth Kit (ATCC, PCS-100-041); All cell lines were confirmed to be mycoplasma negative (MycoAlert Detection Kit, Lonza).

Techniques: Knock-Out, CCK-8 Assay, Migration, Competitive Binding Assay, Flow Cytometry, Labeling, Over Expression, Control, Immunofluorescence, Staining, Cell Culture, Transplantation Assay, Injection, Generated, Fluorescence, Derivative Assay, Isolation

Journal: bioRxiv

Article Title: CXCL-CXCR2 signaling drives cancer-endothelium interactions in SCLC metastatic seeding

doi: 10.64898/2026.04.15.716394

Figure Lengend Snippet: a. Pharmacological inhibition of CXCR2 or RAC1 does not affect SCLC cell viability. Viability of H446, DMS-273, and H82 SCLC cells was quantified via CCK-8 assay following treatment with the CXCR2 inhibitor AZD-5069 ( Upper ) or the RAC1 inhibitor NSC23766 ( Lower ) across the indicated concentration range. No significant changes in relative viable cell number were observed under either treatment condition. b. CXCR2 inhibitor AZD-5069 suppresses migration of adherent SCLC cells. Left: Representative images showing migration of adherent H446 and DMS-273 SCLC cells in the absence or presence of AZD-5069. Right: Migrated SCLC cells per well are quantified and reported as mean ± SD (N = 6 wells). c. RAC1 inhibitor NSC23766 suppresses migration of adherent SCLC cells. Left: Representative images showing migration of adherent H446 and DMS-273 SCLC cells in the absence or presence of NSC23766. Right: Migrated SCLC cells per well are quantified and reported as mean ± SD (N = 6 wells). d. CXCR2 inhibitor AZD-5069 suppresses CXCL-dependent activation of the FAK-RAC-PAK pathway in SCLC cells. Western blot analysis of H82 cells treated for 24 hours with vehicle, CXCLs, AZD-5069, or the combination. CXCL stimulation increased phosphorylation of FAK1 and PAK1, whereas AZD-5069 treatment markedly attenuated CXCL-dependent FAK and PAK phosphorylation. Quantification was performed by intensity fold change from three blots. e. RAC1 inhibitor NSC23766 suppresses CXCL-dependent activation of PAK phosphorylation in SCLC cells. Western blot analysis of H82 cells treated for 24 hours with vehicle, CXCLs, NSC23766, or the combination. CXCL stimulation increased phosphorylation of FAK1 and PAK1, whereas NSC23766 treatment only suppresses CXCL-dependent PAK phosphorylation. Quantification was performed by intensity fold change from three blots. f. CXCR2 inhibitor AZD-5069 suppresses RAC1 reporter activation in H82 cells. RAC1 FRET reporter fluorescence was quantified in H82 cells treated with vehicle or AZD-5069 (N = 15 fields). g. RAC1 inhibitor NSC23766 suppresses RAC1 reporter activation in H82 cells. RAC1 FRET reporter fluorescence was quantified in H82 cells treated with vehicle or NSC23766 (N = 15 fields). h. CXCR2 inhibitor AZD-5069 suppresses F-actin assembly in DMS-273 cells. Left : Representative immunofluorescence images of F-actin staining in DMS-273 cells treated with or without AZD-5069. Right : Quantification of F-actin levels per field, presented as mean ± SD of the target fluorescence to DAPI signal intensity ratio per field (N > 15 fields). i. RAC1 inhibitor NSC23766 suppresses F-actin assembly in DMS-273 SCLC cells. Left : Representative immunofluorescence images of F-actin in DMS-273 cells treated with or without NSC23766. Right : Quantification of F-actin levels per field, presented as mean ± SD of the target fluorescence to DAPI signal intensity ratio per field (N > 15 fields). j. CXCR2 inhibitor AZD-5069 reduces SCLC-endothelial cell interaction strength. GFP transfer from H82 sender to HUVEC receiver cells was quantified via G-baToN assay in the presence or absence of AZD-5069. The percentage of GFP□ cells within the BFP□mCherry□ HUVEC receiver population is presented as mean ± SD (N = 3 cultures). k. RAC1 inhibitor NSC23766 reduces SCLC-endothelial cell interaction strength. GFP transfer from H82 sender to HUVEC receiver cells was quantified via G-baToN assay in the presence or absence of NSC23766. The percentage of GFP□ cells within the BFP□mCherry□ HUVEC receiver population is presented as mean ± SD (N = 3 cultures). l. AZD-5069 treatment suppresses SCLC liver metastatic seeding. MOBA-seq was performed on livers from NSG mice transplanted with a barcoded RP48 SCLC cell library, and metastatic metrics were compared between vehicle and AZD-5069-treated cohorts (N = 5 mice per cohort). Metastatic colonies in the liver are plotted with red diamonds denoting the mean colony size. Histograms show colony size distributions for vehicle and AZD-5069-treated mice. AZD-5069 treatment markedly reduced the number of seeded metastatic colonies without significantly altering mean colony size. m. AZD-5069 treatment suppresses liver metastatic burden. Box plots show the total number of metastatic SCLC cells recovered per mouse liver from vehicle- and AZD-5069-treated cohorts. AZD-5069 treatment significantly reduced total liver metastatic cell number compared with vehicle controls. n. AZD-5069 treatment suppresses liver metastatic seeding. Box plots show the total metastatic colony number recovered per mouse liver from vehicle- and AZD-5069-treated cohorts. AZD-5069 treatment significantly reduced seeded metastatic colony number compared with vehicle controls. o. AZD-5069 treatment prolongs overall survival in metastases-bearing mice. Kaplan–Meier survival curves of mice transplanted with RP48 SCLC cells and treated with vehicle or AZD-5069. AZD-5069 treatment significantly extended overall survival compared with vehicle controls. p. Proposed model of CXCL-CXCR2-RAC1 signaling in cancer-endothelium interactions during SCLC liver metastasis. Circulating SCLC cells engage liver endothelial cells, inducing endothelial CXCL production. CXCLs signal through CXCR2 on SCLC cells to activate RAC1-dependent cytoskeletal remodeling and F-actin assembly, enhancing SCLC migration, adhesion, and sustained interactions with endothelial cells in a positive feedback loop that promotes metastatic colonization. Pharmacological or genetic inhibition of key pathway components, including CXCR2 and RAC1, disrupts this signaling cascade, weakening SCLC-EC interactions and suppressing liver metastasis.

Article Snippet: HUVECs were cultured in Vascular Cell Basal Medium (ATCC, PCS-100-030) with Endothelial Cell Growth Kit (ATCC, PCS-100-041); All cell lines were confirmed to be mycoplasma negative (MycoAlert Detection Kit, Lonza).

Techniques: Inhibition, CCK-8 Assay, Concentration Assay, Migration, Activation Assay, Western Blot, Phospho-proteomics, Fluorescence, Immunofluorescence, Staining