Journal: Cell Communication and Signaling : CCS

Article Title: The aggressive colorectal cancer subtype marker HTR2B has a dual role depending on the tumor microenvironment

doi: 10.1186/s12964-025-02395-6

Figure Lengend Snippet: The CMS4 marker HTR2B shows an intra-tumoral cellular heterogeneity in CRC. A - B Representative confocal microscopic images ( A ) and their quantification ( B ) in PDO lines for CDX2, FRMD6, ZEB1, and HTR2B. C The ratio of single and double positive organoid cells for CDX2 and HTR2B within the CDX2 + or HTR2B + populations (representative confocal image and quantification). D Immunostaining for CDX2 and HTR2B from CRC tissue slides. E The ratio of KI67 + proliferating cells within the CDX2 + or HTR2B + PDO cells (analysis of confocal images). F Immunohistochemistry of tissue slides from three CRC patients for the indicated markers. CDH1 was used as an epithelial cell-specific positive control. Note the epithelial cell restricted heterogeneous expression of HTR2B. G Light microscopic and confocal images from normal human colonic organoids. MUC2 and CHGA are markers of differentiated Goblet and enteroendocrine cells, respectively. Student t-test ( C ) was carried out with ** p < 0.01. Scale bars: 20 μm ( A , C , G ) or 100 μm ( D , F ). n = 4 ( C , E ). For B ), 10–15 organoids were evaluated from each PDO line

Article Snippet: Serotonin, HTR2B agonist (α-methylserotonin), HTR2B inhibitor 1 (RS-127445), HTR2B inhibitor 2 (SB-204741), and Notch inhibitors (γ-secretase inhibitors, DAPT and DBZ, 10 μM, MedChemExpress) were the compounds of interest.

Techniques: Marker, Immunostaining, Immunohistochemistry, Positive Control, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: The aggressive colorectal cancer subtype marker HTR2B has a dual role depending on the tumor microenvironment

doi: 10.1186/s12964-025-02395-6

Figure Lengend Snippet: Unfavorable conditions increase the percentage of HTR2B + cells and lead to a reduced viability of tumor cells induced by serotonin. A Light microscopy images of PDO cultures in control (full), in carbohydrate-free (noGl), and in amino acid-free medium (noAA) and the quantification of relative organoid area compared to the full medium control. The horizontal red line indicates the control with a relative area of 1. B The relative proportion of KI67 + or P-S6 + cells within organoid lines cultured in noGl or noAA medium, compared to full control (horizontal red line). Analysis of confocal images. C The effect of the mTOR inhibitor rapamycin on the ratio of HTR2B + cells under the indicated conditions. For each organoid line, samples were compared to the untreated full medium control (flow cytometry). D Relative RNA level of HTR2B in the presence of rapamycin (1µM, 2 days, RT-qPCR). Values were normalized to the housekeeping gene, and they were then compared to the untreated control (red line). E Changes in the organoid area and the ratio of KI67 + proliferating cells in PDO lines when cultured in full medium for 5 days (analyzed from light and confocal microscopy images). F The effect of different culturing conditions on cell viability, measured by CellTiterGlo 3D. G The effect of serotonin (10µM) on PDO viability compared to untreated samples under different culturing conditions. H Concentration dependent effect of serotonin and agonist, detected by CellTiterGlo 3D test. I Cell viability in the presence/absence of serotonin (10µM), α-methylserotonin (agonist, 10µM), and HTR2B inhibitors (inhibitor 1: RS-1274451, inhibitor 2: SB 204741, both at 1µM) in carbohydrate (noGl) or amino acid-free medium (noAA). J Ratio of HTR2B + cells in the presence or absence of 5-FU. Note that 5-FU was applied at an IC50 concentration for each PDO line that had been determined in our previous studies ( and Table S5). Samples were processed after 3 days of treatment (flow cytometry). K Relative changes in the ratio of P-S6 + cells in the presence of serotonin (10µM) or HTR2B agonist (α-methylserotonin, 10µM). The horizontal line indicates the control (flow cytometry). L Relative cell viability in the presence of the indicated treatments, compared to control samples (CellTiterGlo 3D analysis). Scale bars: 50 μm. Paired ( A - G and K ), unpaired t-test ( J ) or ANOVA with Tukey post hoc test ( H , I , L ) were applied with * p < 0.05, ** p < 0.01, *** p < 0.005, ns: p > 0.05. n = 4 (exception: n = 3 for B)

Article Snippet: Serotonin, HTR2B agonist (α-methylserotonin), HTR2B inhibitor 1 (RS-127445), HTR2B inhibitor 2 (SB-204741), and Notch inhibitors (γ-secretase inhibitors, DAPT and DBZ, 10 μM, MedChemExpress) were the compounds of interest.

Techniques: Light Microscopy, Control, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Confocal Microscopy, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: The aggressive colorectal cancer subtype marker HTR2B has a dual role depending on the tumor microenvironment

doi: 10.1186/s12964-025-02395-6

Figure Lengend Snippet: Fibroblasts induce a partial EMT and an increased HTR2B expression in CRC cells. A Light microscopy images from 3D cultures of fibroblasts, PDOs, or their co-cultures. B Confocal microscopic image of a co-culture. Fibroblasts were labeled with the membrane staining dye DiD before co-culturing (3D reconstruction, background immunostaining for collagen-IV). Note the close proximity of fibroblasts and organoids. C Analysis of cell surface EpCAM level from cultures containing only organoids or co-cultures with fibroblasts (representative flow cytometry dot plots and their quantification). Note that EpCAM is an epithelial cell marker. Isotype control samples were used to determine the unspecific background (green line). D Relative ratio of LUM or HTR2B positive cells in PDOs and co-cultures when gating for the EpCAM + cells in flow cytometry. Note that fibroblasts do not express the epithelial marker EpCAM. E Immunostaining of CRC tissue sections for the indicated markers. Scale bars: 50 μm ( A ), 20 μm ( B ), 25 μm ( E ). Paired ( C ) or unpaired ( D ) t-tests were used with * p < 0.05, ** p < 0.01. n = 3 ( D ) or n = 4 ( C )

Article Snippet: Serotonin, HTR2B agonist (α-methylserotonin), HTR2B inhibitor 1 (RS-127445), HTR2B inhibitor 2 (SB-204741), and Notch inhibitors (γ-secretase inhibitors, DAPT and DBZ, 10 μM, MedChemExpress) were the compounds of interest.

Techniques: Expressing, Light Microscopy, Co-Culture Assay, Labeling, Membrane, Staining, Immunostaining, Flow Cytometry, Marker, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The aggressive colorectal cancer subtype marker HTR2B has a dual role depending on the tumor microenvironment

doi: 10.1186/s12964-025-02395-6

Figure Lengend Snippet: Activating HTR2B induces invasion of PDO cells in collagen-I. A Morphology of PDOs in Matrigel and collagen-I (left panel) and the relative invasion area in collagen-I (right panel). For comparison, the horizontal line indicates Matrigel control. For measuring invasion area, see Materials and Methods. B RNA level of the indicated genes in PDOs cultured in collagen, compared to Matrigel (red line) (RT-qPCR). C Relative ratio of VIM+, LUM + or HTR2B + cells when cultured in collagen-I, compared to Matrigel (representative confocal microscope images and their quantification). D Ratio of HTR2B + cells in the invasion zone compared to the organoid core (confocal microscopy, collagen-I cultures, representative image, and statistical evaluation). The yellow line shows the segmentation between the organoid core and the invasion zone. E The effect of serotonin or agonist (10µM) on the proportion of VIM + cells (analysis of confocal microscopy images). F The effect of HTR2B agonist on the ratio of LUM + and LUM+/VIM + double positive cells in the organoid lines. G Changes in the invasion area in collagen-I. H The effect of serotonin and HTR2B agonist compared to control samples in collagen, measured by CellTiterGlo 3D. I The ratio of dead cells in the presence of the indicated treatments in collagen (cell viability test: LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit and flow cytometry). J The proportion of the apoptotic active caspase-3 + cells in collagen (flow cytometry). K Viability in the presence of the indicated treatments in collagen-I (CellTiterGlo 3D assay). Scale bars: 50 μm ( A ), 25 μm ( C - D ). Paired ( A - H ), unpaired t-tests ( I - J ) or ANOVA with Tukey post hoc test ( K ) were applied with * p < 0.05, ** p < 0.01, *** p < 0.005. n = 4 ( n = 3 for G)

Article Snippet: Serotonin, HTR2B agonist (α-methylserotonin), HTR2B inhibitor 1 (RS-127445), HTR2B inhibitor 2 (SB-204741), and Notch inhibitors (γ-secretase inhibitors, DAPT and DBZ, 10 μM, MedChemExpress) were the compounds of interest.

Techniques: Comparison, Control, Cell Culture, Quantitative RT-PCR, Microscopy, Confocal Microscopy, Staining, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: The aggressive colorectal cancer subtype marker HTR2B has a dual role depending on the tumor microenvironment

doi: 10.1186/s12964-025-02395-6

Figure Lengend Snippet: HTR2B +/high tumor cells have a higher invasion potential compared to the HTR2B -/low population. A Representative flow cytometry histogram and the sorting strategy. B - C Ratio of HTR2B+ ( B ) or KI67 + proliferating cells ( C ) in the organoids derived from sorted HTR2B +/high and HTR2B -/low PDO cells, analyzed after 7 days in Matrigel (confocal microscopy). D The area of organoids from sorted cells (light microscopic images, analysis with ImageJ). E RNA level of the indicated genes in HTR2B +/high cell derived organoids compared to the HTR2B -/low organoids (RT-qPCR). F VIM + and LUM + cells in the HTR2B +/high cell-derived organoids compared to HTR2B -/low organoids (marked with a horizontal line). Analysis was carried out after 7 days in culture (confocal microscopy). G Changes in the indicated parameters of HTR2B +/high organoids compared to the HTR2B -/low ones (light microscopic image analysis). H The effect of serotonin and HTR2B agonist (10 μM) on HTR2B +/high or HTR2B -/low cell-derived organoids (light microscopy, measured after 7 days in collagen-I, compared to the respective untreated control). Organoid area and invasion area were measured. I Relative RNA level of the indicated genes in HTR2B +/high organoids compared to HTR2B -/low PDOs (RT-qPCR). Expression values were first normalized to housekeeping, and then the value from HTR2B -/low PDOs was taken as 1 (horizontal line). J NOTCH3 signal intensity in the sorted HTR2B +/high cells compared to the HTR2B -/low population (flow cytometry). Unpaired (B-D) or paired ( E - J ) t-tests were used with * p < 0.05, ** p < 0.01, *** p < 0.005, ns: p > 0.05. n = 4

Article Snippet: Serotonin, HTR2B agonist (α-methylserotonin), HTR2B inhibitor 1 (RS-127445), HTR2B inhibitor 2 (SB-204741), and Notch inhibitors (γ-secretase inhibitors, DAPT and DBZ, 10 μM, MedChemExpress) were the compounds of interest.

Techniques: Flow Cytometry, Derivative Assay, Confocal Microscopy, Quantitative RT-PCR, Light Microscopy, Control, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: The aggressive colorectal cancer subtype marker HTR2B has a dual role depending on the tumor microenvironment

doi: 10.1186/s12964-025-02395-6

Figure Lengend Snippet: NOTCH3 +/high tumor cells share common features with HTR2B +/high cells in a permissive microenvironment. A RNA level of NOTCH1 and NOTCH3 in four PDO lines, normalized first to housekeeping and then to AXIN2 (RT-qPCR). Note that the Wnt target gene AXIN2 (used for comparison) is expressed at a high level in CRC cells. B RNA level of NOTCH1 and NOTCH3 in collagen compared to Matrigel (RT-qPCR). Data were normalized to housekeeping control, and then Matrigel values were taken as 1. C Expression of the indicated genes in PDO lines. Note that data were compared first to housekeeping and then to NOTCH3 to illustrate NOTCH ligand levels relative to their receptor. D RNA level of NOTCH ligands in fibroblasts (CAF, n = 6) compared to organoids ( n = 4, RT-qPCR). E Flow cytometry histogram of cell surface NOTCH3 level from two different PDO lines. F - G ) The effect of the NOTCH inhibitor DAPT and DBZ (10 µM) on the organoid ( F ) and invasion area ( G ) in collagen-I (light microscopy, image analysis by ImageJ). Note that organoids grown in Matrigel for 4 days were embedded into collagen-I for a further 5 days. H Relative invasion area (compared to control) in the presence of the indicated treatments. I Representative confocal microscopy images and their quantification for KI67. NOTCH3 +/high and NOTCH3 -/low cells were sorted, and organoids were analyzed on day 7. J Relative area of NOTCH3 +/high organoids compared to NOTCH3 -/low cell-derived organoids (light microscopy). K Fold change in the RNA level of the indicated genes in the NOTCH3 +/high compared to NOTCH3 -/low organoids (RT-qPCR). Expression data were first normalized to housekeeping, and then values of NOTCH3 -/low samples were taken as 1. L Flow cytometry from the tumor tissues of two CRC patients. Note that signals for HTR2B and NOTCH3 were analyzed from EpCAM + cells. M - O Relative RNA of the indicated genes in the presence of DAPT ( M , 10µM), the HTR2B agonist ( N , 10µM) or rapamycin ( O , 1 µM). RT-qPCR data were normalized to housekeeping and then compared to the untreated controls (red line). Scale bars: 20 μm. Unpaired ( D ) or paired ( A - C , E - O ) t-tests were applied with * p < 0.05, ** p < 0.01 and *** p < 0.005, ns: p > 0.05. n = 3–4

Article Snippet: Serotonin, HTR2B agonist (α-methylserotonin), HTR2B inhibitor 1 (RS-127445), HTR2B inhibitor 2 (SB-204741), and Notch inhibitors (γ-secretase inhibitors, DAPT and DBZ, 10 μM, MedChemExpress) were the compounds of interest.

Techniques: Quantitative RT-PCR, Comparison, Control, Expressing, Flow Cytometry, Light Microscopy, Confocal Microscopy, Derivative Assay