keratinocyte growth supplement  (Thermo Fisher)

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: a , Study schema depicting the discovery and validation phase (created with BioRender.com ). We applied scRNA-seq, RNAscope, novel PNI organoids, and survival analysis to define the tumor cellular niche and therapeutically actionable intercellular pathways of PNI pos cancers. b , Uniform manifold approximation and projection (UMAP) of the cSCC cellular atlas, including PNI-positive and PNI-negative tumors compared to normal skin (n=92,371). c , UMAP view of epithelial cells, fibroblasts, mast cells, T cells, B/Plasma cells, and melanocytes. d , UMAP plot for keratinocytes in PNI pos cSCC (n=4830). e , UMAP of different keratinocyte subpopulations involved in PNI pos cSCC.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques: Biomarker Discovery, RNAscope, Clinical Proteomics

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: a , UMAP view of myeloid, endothelial, and parotid cells in cSCC. b , DvP score depicting the loss of differentiation programming in keratinocytes in PNI pos cSCC. P-value was calculated by mixed effect model adjusting for patient ID with the Satterthwaite method. c , UMAP of basal keratinocytes in PNI pos cSCC. d , Gene set enrichment analysis (GSEA) for a stress-like signature in TCGA tumors with known PNI status. P-value was calculated by permutation test.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: a , Left, circle plot analysis displaying cellular crosstalk via ligand ( KITLG ) expressed by keratinocytes/fibroblasts and receptor ( KIT ) present on mast cells in PNI pos cSCC. Right, scRNA-seq data for KITLG and KIT expression in different cellular populations of PNI pos cSCC. b , Expression of KITLG in keratinocytes and fibroblasts in cSCC patients. Wilcoxon rank sum test calculated the P-value . c , Enrichment of KITLG and mast cell infiltration gene signature in TCGA tumors with known PNI status. P-value calculated by linear regression after adjusting for cancer types. Error lines represent 1.5X interquartile range, and the box represents the interquartile range. d , RNAscope duplex in situ hybridization of KITLG (teal), KIT (red), and counterstained cell nuclei (blue) in normal skin, PNI neg , and PNI pos cSCC. Scale bar=100μm, images are representative of n=5/group. e , RNAscope duplex in situ hybridization of KITLG (teal), KIT (red), and counterstained cell nuclei (blue) in normal biliary epithelium, PNI neg , and PNI pos CHOL. Scale bar=100μm, images are representative of n=5/group. f , RNAscope duplex in situ hybridization of KITLG (teal), KIT (red), and counterstained cell nuclei (blue) in normal oral mucosa, PNI neg , and PNI pos HNSC. Scale bar=100μm, images are representative of n=5/group.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques: Expressing, RNAscope, In Situ Hybridization

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: a , Copy number analysis of KITLG in primary human cSCC. Fisher’s exact test calculated P-value . b , Single-cell regulatory network inference and clustering (SCENIC)-based prediction of upstream regulators of KITLG in keratinocytes. Error lines represent 1.5X interquartile range, and the box represents the interquartile range. c , Pathway activity scores of predicted KITLG regulators in PNI neg cSCC and KITLG -amplified and KITLG -diploid PNI pos tumors. Error lines represent 1.5X interquartile range, and the box represents the interquartile range. d , IGV view of KITLG gene body with open chromatin, transcription regulators, and transcription factor binding analyzed using publicly available ChIP-seq data. e , Expression analysis of CXCL10 and KITLG upon STAT1 phosphorylation inhibition with fludarabine in keratinocytes. Conducted with replicates and repeated four independent times. Two-tailed Student’s t-tests calculated the P-value , error bar-standard deviation.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques: Activity Assay, Amplification, Binding Assay, ChIP-sequencing, Expressing, Phospho-proteomics, Inhibition, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: a , Expression of various mast cell mediator receptors in keratinocyte populations of PNI neg and PNI pos cSCC assessed by scRNA-seq. Wilcoxon rank sum test calculated the P-value . b , Expression analysis of HRH1 in PNI-annotated TCGA tumors. P-value calculated by linear regression after adjusting for cancer types. Error lines represent 1.5X interquartile range, and the box represents the interquartile range. c , Release of histamine by mast cells investigated with toluidine blue at low pH in normal skin, PNI neg, and PNI pos cSCC. Deep purple-histamine-positive cells, light blue-background, scale bar=100μm, images are representative of n=5/group. d , Release of histamine by mast cells in normal biliary epithelium, PNI neg, and PNI pos CHOL. Deep purple-histamine-positive cells, light blue-background, scale bar=100μm, images are representative of n=5/group. e , Release of histamine by mast cells in normal oral mucosa, PNI neg, and PNI pos HNSC. Deep purple-positive cells, light blue-background, scale bar=100μm, images are representative of n=5/group. f , HRAS and CDK4 -transformed tumor spheroids were treated with either vehicle control, histamine, or a combination of histamine and H1-specific or H2-specific antihistamine. Tumor spheroid invasion was evaluated with live (green) and dead (red) staining and fluorescence microscopy. Scale bar=100μm. Results are representative of three independent experiments conducted in replicates. g , Immunofluorescence analysis of PNI organoids treated with either vehicle, histamine, or a combination of histamine and H1-specific or H2-specific antihistamine. The dotted line outlines the nerve sheath. Scale bar=100μm. Results are representative of three independent experiments conducted in replicates.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques: Expressing, Transformation Assay, Control, Staining, Fluorescence, Microscopy, Immunofluorescence

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: a , Quantification of histamine-positive mast cells using toluidine blue in PNI neg and PNI pos cSCC, CHOL, and HNSC. A three-five field of view was used to quantify histamine-positive cells from images representing n=5/group. b-c , The Dose response of histamine treatments in keratinocytes was evaluated with HRH1 expression via qRT-PCR and immunoblotting. Conducted with replicates and repeated three independent times. Two-tailed Student’s t-tests calculated the P-value , error bar-standard deviation, Hist-histamine. d , Immunoblotting for P-JNK, JNK, and β-Actin upon histamine treatment in primary keratinocytes. Repeated three times. e , qRT-PCR analysis of HRH1 in different doses of H1-or H2-antihistamines following histamine treatment in keratinocytes compared to histamine group. Conducted with replicates and repeated three independent times. Two-tailed Student’s t-tests calculated the P-value , error bar-standard deviation, Hist-histamine, Niza-Nizatidine, CF-clemastine fumarate, Des-Desloratadine.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: Oncogenic HRAS and CDK4 -transformed primary keratinocytes were grown on human dermis at the air-liquid interface for 14 days and subjected to the drug treatments as shown. Immunofluorescence staining of keratin 5 (red), col VII (green), and nuclei (blue) was performed on the resulting cSCC organoids. Niza-Nizatidine, CF-clemastine fumarate, Des-Desloratadine. Scale bar=100μm, conducted with replicates and repeated three independent times.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques: Transformation Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

doi: 10.1101/2025.06.23.661147

Figure Lengend Snippet: a , Phosphoproteomic analysis of keratinocytes treated with either vehicle control or histamine for 10 minutes by mass spectrometry. The heatmap highlights the phosphorylation sites altered by histamine compared to the control. b , Pathway enrichment analysis by EnrichR of histamine phosphorylated sites in primary keratinocytes. c , Left, P38 gene signature enrichment in PNI-annotated TCGA tumors. Right, induction of various MMPs in PNI-positive TCGA tumors. P-value calculated by linear regression after adjusting for cancer types. Error lines represent 1.5X interquartile range, and the box represents the interquartile range. d , Immunoblotting of P-P38, P38, and β-Actin in primary keratinocytes treated with vehicle control, hist-histamine, SB-SB203580 (P38 inhibitor) with histamine, or CF-clemastine fumarate (H1-antihistamine) with histamine. Results are representative of at least three independent experiments. e , Left, expression analysis of MMPs in primary keratinocytes treated with control, hist-histamine, SB-SB203580 (P38 inhibitor) with histamine, or CF-clemastine fumarate (H1-antihistamine) with histamine. Results are representative of at least three independent experiments conducted in replicates. Two-tailed Student’s t-tests calculated the P-value by comparing Hist to control, then CF and SB to the Hist group. Error bar-standard deviation. Right, ELISA for MMP2 and MMP9 in culture supernatants of primary keratinocytes in different treatment conditions. hist-histamine, SB-SB203580, CF-clemastine fumarate. Results are representative of multiple replicates in an experiment. Two-tailed Student’s t-tests calculated the P-value by comparing Hist to control, then CF and SB to the Hist group. Error bar-standard deviation. f , Live (green) and dead (red) staining in spheroids depicted tumor spheroid invasion upon the vehicle, histamine, or P38 inhibitor (SB203580) and histamine treatments. Scale bar=100μm, Results are representative of three independent experiments conducted in replicates. g , Immunofluorescence of Keratin 5 (red) displaying nerve invading oncogenic HRAS and CDK4 -transformed primary keratinocytes in PNI organoids treated with histamine, or P38 inhibitor (SB203580) and histamine. The dotted line outlines the nerve sheath. Scale bar=100μm, Results are representative of three independent experiments conducted in replicates.

Article Snippet: Cells were maintained at 37°C with 5% CO2 in medium 154 (Thermo Fisher Scientific) supplemented with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific).

Techniques: Control, Mass Spectrometry, Phospho-proteomics, Western Blot, Expressing, Two Tailed Test, Standard Deviation, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Transformation Assay