Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Manipulating NGF affects P2X3 receptor-mediated currents. A, Representative examples of currents induced by α,β-meATP (black bar) on TG neurons cultured in control condition (left), after treatment with anti-NGF antibody (24 h, middle) or incubated with NGF (50 ng/ml, 24 h). B, Dose–response curves for α,β-meATP in control condition (filled circles; n = 16–24), after anti-NGF antibody treatment (filled triangles; n = 8–24; *p = 0.03), and after NGF treatment (open circles; n = 12; *p = 0.033). Current amplitudes (ΔIN) were normalized with respect to the value obtained with 10 μm α,β-meATP; p values were calculated with absolute values. Note that, despite the effect of NGF or anti-NGF antibody application on α,β-meATP-induced current amplitude, there is no difference in agonist EC50 values. C, Histogram of real-time RT-PCR experiments of TG culture mRNAs shows that NGF increases P2X3 neosynthesis (n = 3 experiments; *p = 0.03). D, Example of Western immunoblots of P2X3 receptor from total extracts of TG neurons in control (lane 1), in anti-NGF antibody (24 h; lane 2), or NGF (50 ng/ml, 24 h; lane 3). Bottom lanes show control loading with β-tubulin III. Histograms show P2X3 optical density values expressed in AUs in the different culture conditions normalized with respect to the β-tubulin III signal (mean values in AUs; n = 5 experiments; p > 0.05).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Cell Culture, Incubation, Quantitative RT-PCR, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Manipulating NGF affects desensitization of currents induced by α,β-meATP. A, Representative examples of currents induced by applications of α,β-meATP (black bar) in control condition (left), after anti-NGF antibody treatment (middle), and after NGF treatment (right). Traces of 10 μm α,β-meATP-evoked currents recorded after a second agonist application (30 s interval; see arrows) are superimposed. Note the differential recovery from desensitization in the three conditions. B, Histograms show no change in the time constant values of the α,β-meATP-evoked current decay, used as an index of desensitization onset (τfast; n = 151, n = 56, and n = 46 for control, anti-NGF, and NGF, respectively). C, Histograms show that anti-NGF treatment reduces recovery (*p < 0.0001) from desensitization of P2X3-mediated currents, whereas NGF increases (*p < 0.001) this value (n = 99, n = 49, and n = 38 for control, anti-NGF, and NGF, respectively). D, Representative traces of Ca2+ transients show that, with paired α,β-meATP application (black dots, 30 s spaced), the second response is more depressed after anti-NGF treatment than in control. Neurons are identified by their responsiveness to KCl (50 mm, 1 s), which remains unchanged after anti-NGF treatment. E, Histograms showing the depressant action of anti-NGF treatment on the amplitude of Ca2+ transients induced by 10 μm α,β-meATP or 50 mm KCl. Control level is indicated by a dashed line. *p ≤ 0.05; n = 4–5 experiments (30–40 cells in each experiment).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Phosphorylation state of P2X3 receptors is controlled by NGF. A, Example of P2X3 receptor immunoprecipitation (IP) detected in Western blot (WB) with anti-phospho-threonine antibody. P2X3 receptors from untreated TG cultures show constitutive threonine phosphorylation (lane 1). Anti-NGF-treated samples show a reduced level of P2X3 threonine phosphorylation (lane 2), which is restored after acute application of NGF (50 ng/ml, <15 min) (lane 3). Total P2X3 inputs derived from the same lysates and immunostained with anti-P2X3 antibody are also shown (bottom lanes). B, Histograms show mean values (optical density AUs) of phosphorylated P2X3 subunits obtained from anti-NGF (*p= 0.008; n = 8 experiments) or anti-NGF plus NGF (*p= 0.009; n = 8) experiments. C, Acute enhancement by NGF (50 ng/ml) of α,β-meATP-induced currents (top records) is prevented by the PKC inhibitor chelerythrine (1.5 μm in the patch pipette; bottom records). D, Histograms of changes in P2X3 subunit threonine phosphorylation after acute application of NGF (*p = 0.036; n = 9)

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Immunoprecipitation, Western Blot, Derivative Assay, Transferring

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Anti-NGF treatment increases expression of heteromeric P2X2/3 receptors. A, Histograms showing residual current amplitude in control (n = 187) and after anti-NGF (n = 59) or NGF (n = 70) treatment. *p < 0.05. B, Cumulative probability plot of the ratio Iresidual/Ipeak for control condition (filled circles; n = 224), anti-NGF antibody treatment (filled triangles; n = 79), and NGF treatment (open circles; n = 85). Note that antibody-treated, but not NGF-treated, neurons show larger Iresidual, indicative of higher contribution by heteromeric P2X2/3 receptors. The activity of heteromeric P2X2/3 receptors is estimated as the ratio between the amplitude of the steady-state Iresidual at the end of α,β-meATP application and the one of the peak current (Ipeak). C, Example of biotinylation experiments showing that NGF deprivation treatment increases the surface expression of the P2X2 subunit (top), although it does not affect the amount of surface P2X3 (bottom). Histograms indicate changes in P2X2 (black) or P2X3 (white) surface receptors measured with optical density values expressed in AUs (n = 6 or 3 experiments, respectively; *p = 0.005) and normalized with respect to the protein amount in control condition (dashed line). D, Confocal microscopy photographs of TG neurons in the control and after anti-NGF treatment show different distribution of P2X2 immunostaining. In the absence of NGF, TG neurons express P2X2 receptors on the cell surface or close by (2.5 ± 1.5 μm beneath the surface; n = 15). Scale bar, 10 μm. Histograms show the relative percentage of neurons showing diffused or ring-like P2X2 immunoreactivity in control or after anti-NGF treatment (n = 469 or 211, respectively; *p < 0.001). E, Immunopurification of heteromeric P2X2/3 receptors was obtained with chemical cross-linking treatment (DTSSP) of membrane proteins. TG neuron extracts are immunoprecipitated (IP) with anti-P2X3 antibody and immunoblotted using anti-P2X2 antibody (lanes 1, 2). P2X2 subunit (64 kDa) is immunopurified from anti-NGF antibody-treated extracts (lane 2, arrowhead) and not from control (lane 1; n = 5 experiments) after DTSSP cross-linking. Anti-NGF-treated extracts, without DTSSP cross-linking, do not show any P2X2 signal (lane 3). P2X2 immunoprecipitation detects a single band (performed as control; lane 4, arrowhead). WB, Western blot. F, Anti-NGF treatment increases the fraction of TG neurons double immunopositive for P2X2 and P2X3 subunits (*p = 0.023), whereas chronic NGF application does not change this value with respect to control (n = 4).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Expressing, Activity Assay, Confocal Microscopy, Immunostaining, Immu-Puri, Immunoprecipitation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: The potentiating action of NGF on P2X3 receptor-mediated responses does not depend on CGRP. A, Examples of currents induced by α,β-meATP in TG neurons grown in control condition, in the presence of NGF (50 ng/ml, 24 h), in the presence of CGRP receptor antagonist BIBN4096BS (BIBN; 50 ng/ml, 24 h), or with NGF plus BIBN4096BS (24 h). B, Somatic size distribution of TG neurons immunostained with TrkA receptor antibody (filled columns) and labeled with CGRP-RITC (open columns) to investigate colocalization of NGF and CGRP receptors (filled, cross-hatched columns). Data are from ∼1000 cells (n = 3 independent experiments). IR, Immunoreactivity. C, Example of patch-clamp traces showing that CGRP application (1 μm, 1 h) to anti-NGF antibody-treated neurons increases α,β-meATP responses and accelerates recovery from desensitization (30 s interval between twin pulses). D, CGRP (1 μm, 1 h) potentiates α,β-meATP-evoked currents in control condition (n = 19; *p < 0.0001) and after anti-NGF treatment (n = 18; *p < 0.0001).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Labeling, Patch Clamp

Journal: The Journal of Neuroscience

Article Title: Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X 3 Receptors of Nociceptive Trigeminal Ganglion Neurons

doi: 10.1523/JNEUROSCI.0713-07.2007

Figure Lengend Snippet: Trigeminal nociception evoked by α,β-meATP is prevented by anti-NGF treatment. A, Histograms showing the time spent by mice in face-rubbing activity after subcutaneous injection of α,β-meATP (10 μl bolus of 10 mm solution) into the upper lip after pretreatment with anti-NGF or saline (sal). Each bar is the mean of six to eight animals observed for the first 15 min epoch. No significant changes in rubbing activity are observed during this period. All mice were pretreated 24 h earlier with saline (10 μl/g, i.p.) or anti-NGF antibody (300 ng/g, i.p.). B, In the 15–30 min epoch, a significant increase (*p = 0.005) in rubbing activity is observed after injection of α,β-meATP (n = 8). This algogenic effect of α,β-meATP is blocked (*p= 0.025 vs sham pretreatment) after anti-NGF pretreatment (n = 7).

Article Snippet: The following substances were added to the culture medium as required: NGF (50 ng/ml; Alomone Labs, Jerusalem, Israel), neutralizing anti-NGF-2.5S, -β, and -7S antibody [from male mouse submaxillary glands, 6 μg/ml, 1:5000 (Sigma, Milan, Italy) or 1:1000 (Chemicon, Hampshire, UK)], the nonpeptide CGRP receptor antagonist BIBN4096BS [1-piperidinecarboxamide, N -[2-[[5-amino-L-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2 H )-quinazolinyl)] (50 n m ; a gift from Boehringer Ingelheim, Ingelheim, Germany), CGRP (1 μ m ; Sigma), and chelerythrine chloride (5 μ m ; Sigma).

Techniques: Activity Assay, Injection

Journal: The Journal of Neuroscience

Article Title: Self-Complementary AAV9 Gene Delivery Partially Corrects Pathology Associated with Juvenile Neuronal Ceroid Lipofuscinosis (CLN3)

doi: 10.1523/JNEUROSCI.1635-16.2016

Figure Lengend Snippet: Comparative biodistribution of scAAV9 constructs in the Cln3Δex7/8 brain after systemic delivery. One-month-old Cln3Δex7/8 mice (n = 4–5/group) received 1 intravenous injection of 2 × 1012 vg of scAAV9/MeCP2-GFP or scAAV9/β-actin-GFP, whereupon animals were killed at 5 months after injection for analysis of virus biodistribution. A, Quantitation of total percentage GFP+ cells in the S1BF, striatum (STR), suprachiasmatic nucleus (SCN), thalamus (VPM/VPL), hippocampus (CA1/CA3), and VC. B, Quantification of GFP+ neurons (NeuN+GFP+) and astrocytes (GFAP+GFP+) in the S1BF, TH, and VC of scAAV9/β-actin-GFP vs scAAV9/MeCP2-GFP treated mice. C, Tiled 40× confocal images depicting preferential GFP expression in NeuN+ neurons with scAAV9/MeCP2-GFP compared with scAAV9/β-actin-GFP, with both constructs targeting astrocytes. Significant differences are denoted by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001 using a paired t test).

Article Snippet: To quantify transgene expression in various CNS cell types, colocalization of transgene-positive cells (GFP + ) with NeuN + neurons and GFAP + astrocytes was determined using the Imaris program (Bitplane).

Techniques: Construct, Injection, Virus, Quantitation Assay, Expressing

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Schematic of metastatic LUAD and patient-derived xenograft (PDX) engraftment. b , Box and whisker plots of L1CAM IHC H-score in patient-derived primary tumor and metastasis samples. Primary tumor, n = 15; metastases, n = 54. *** P = 0.0002. c , Box and whisker plots of L1CAM IHC H-score in the primary tumors in ( b ) and in PDXs derived from primary tumor or metastases. Primary tumors, n = 15; primary tumor-derived PDXs, n = 36; metastasis-derived PDXs, n = 70. ns, P = 0.8811; * P = 0.0294 (left), 0.0420 (right). d , Schematic of the oncogenic transformation of AT2 cells into lung adenocarcinoma in the KP GEMM ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the KP mouse lungs and tumor areas at different time points after lentivirus instillation ( right panel ). Week 0, n = 6; week 14-19, n = 6; week 20-32, n = 15. ns, P = 0.5710; ** P = 0.0032. e , Schematic of the tumor tissue section ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the tumor center and invasive front ( right panel ). Tumor center, n = 300 cells; invasive front, n = 258 cells. N = 7 image frames. * P = 0.0152. f , L1CAM immunofluorescence (IF) staining of LUAD patient tissue samples at the tumor center (P, papillary) or the invasive front (M, micropapillary). Magnified regions are indicated in red boxes. Scale bar, 20 μm. g , Confocal microscopy image of L1CAM IF staining and Hoechst counterstaining of nuclei in KP tumoroids grown for 5 days. Schematic of tumoroids generated from KP-derived cancer cells ( lower right panel ). Scale bar, 10 μm. h , Percentage of L1CAM + cells in KP primary tumors versus tumoroids. Primary tumor, n = 4 experiments; tumoroids, n = 6 experiments. Mean ± s.e.m. ** P = 0.0095. i , Widefield fluorescence microscopy image of KP tumoroids stained with calcein AM in KP tumor cells sorted by L1CAM expression and grown as tumoroids for 7 days. The magnified region showing a single cell is indicated by a dotted box and that of a tumoroid by a solid box. Scale bar, 400 μm. j , Box and whisker plots of tumoroids formed per 1,000 cells after 7 days in culture. L1CAM − , n = 16; L1CAM + , n = 16. **** P < 0.0001. k , Box and whisker plots of cross-section area per tumoroid in the experiment of panel ( I ). L1CAM - , n = 807; L1CAM + , n = 477. **** P < 0.0001. l , Schematic representation of the generation of L1cam knockout KP LUAD GEMMs. m , H&E staining of KP and KPL1 primary tumors (26 week post-Cre) followed by their histopathological grading using an automated deep neural network. Magnified regions are shown in a red square. Scale bar, 1 mm. n , Fraction of KP and KPL1 tumors (22-26 week post-Cre) based on histopathological grading. Grade 1, green ; Grade 2, orange ; Grade 3, blue ; Grade 4, red . KP, n = 4; KPL1, n = 4. Mean ± S.D. o , H&E staining of KP metastasis in the subcapsular sinus of the lymph node (top panel) or ribcage bone (bottom panel). Invasive front is shown with a dotted yellow line. T, tumor; LN, lymph node. Scale bar, 100 μm. p , Incidence of primary tumor and spontaneous metastases upon viral transduction in KP and KPL1 mice. The organ specificity of spontaneous metastasis is shown as a percentage of the total metastatic tumors. LN, lymph node. KP primary, n = 46; KPL1 primary, n = 30; KP met, n = 42; KPL1 met, n = 16. ns, P = 1; * P = 0.0122. q , Representative images of subcutaneous tumors formed 6 weeks after inoculating 500 KP or KPL1 cells in athymic mice. Scale bar, 2 mm. r , Limiting dilution assay of subcutaneous tumor formation at various doses of KP or KPL1 cells in athymic mice. n = 10 for each condition. s , Fluorescent images of GFP + KP or KPL1 cells seeded in lungs at week 1 after tail vein injection (10 5 cells each) into athymic mice. Scale bar, 10 μm. t , Quantification of KP and KPL1 cells seeded in lungs from the experiment in ( S ). KP, n = 9; KPL1, n = 10. ns, P = 0.3154. u , Representative image of ex vivo lung BLI at week 5 after tail vein injection of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. v , Quantification of ex vivo lung BLI signal in the experiment in ( u ). KP, n = 28; KPL1, n = 14. *** P = 0.0006. w , The KM plot showing the overall survival after performing tail vein injections with KP or KPL1 cells. KP, n = 10; KPL1, n = 10. **** P < 0.0001. Statistical significance was assessed using the two-tailed Mann-Whitney test ( b , e , h , j , k , n , t , v ), one-way analysis of variance followed by the Tukey test ( c , d ), two-tailed Fisher’s exact test ( p ) or log-rank (Mantel-Cox) test ( o , w ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( b - e , j , k , t , v ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Derivative Assay, Whisker Assay, Transformation Assay, Immunofluorescence, Staining, Confocal Microscopy, Generated, Fluorescence, Microscopy, Expressing, Knock-Out, Transduction, Limiting Dilution Assay, Injection, Ex Vivo, Suspension, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Representative L1CAM IHC staining in primary tumors, pleural fluids, and metastatic samples from LUAD patients and three tissue microarrays (Array 1, green; Array 2, blue; Array 3, red) containing normal human lung tissue and LUAD PDX (Passage 0 or 1). Scale bar, 100 μm. b , Heatmap of L1CAM IHC staining quantification (H-score) in primary tumor, pleural fluid, metastatic samples, and PDXs from LUAD patients. Each column represents an individual sample. Samples are color-coded according to their site of tissue. c , Maximum projection of a PDX tumoroid fluorescently stained with L1CAM. Scale bar, 10 μm. d , Cross-section of a PDX tumoroid stained with L1CAM IF and DAPI (nuclei). Scale bar, 10 μm. e , Close up of a PDX tumoroid stained with L1CAM IF and DAPI. Scale bar, 5 μm.

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , L1CAM IHC staining in lung and tumor sections in KP mice at baseline and at different time points after lentivirus instillation to induce adenocarcinoma formation. Yellow, week 0; orange, week 14; red, week 24. Scale bar, 50 μm. b , L1CAM IHC staining of the patient tissue sections including normal tissue and tumor. The invasive tumor front is indicated with a yellow line. Scale bar, 200 μm. c , H&E and L1CAM IHC staining of KP primary tumor sections (26 week post-Cre) showing the indicated histological subtypes. Scale bar, 50 μm. d , Fraction of histological subtypes observed in KP primary tumors. n = 203,870 cells. e , Percentage of L1CAM + cells in KP primary tumors with respect to histological subtypes. Lepidic ( n = 26,871); Papillary ( n = 88,381); Micropapillary (red colored, n = 4,116); Acinar ( n = 3,430); Solid ( n = 81,072). **** P < 0.0001. f , Representative GFP (cancer cells) IF and L1CAM IF images of primary tumor and an autochthonous lymph node metastasis from a KP mouse (35 week post-Cre). Scale bar, 20 μm. g , Quantification of L1CAM IF intensity in the experiment ( f ). LN, lymph node; AU, arbitrary unit. n = 24 image frames; N = 3 mice in each condition. **** P < 0.0001. h , H&E staining of KP tumoroids grown in Matrigel over the course of 7 days. Scale bar, 100 μm. I , Brightfield and GFP fluorescence microscopy images of KP tumoroids at day 7. Scale bar, 10 μm. j , Close up of L1CAM IHC staining of patient samples and KP tumors. Scale bar, 20 μm. Statistical significance was assessed using the two-tailed Mann-Whitney test ( g ) or one-way analysis of variance followed by the Tukey test ( e ). Data are shown as mean ± S.D. ( d , e ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining, Fluorescence, Microscopy, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Representative images of tumor-bearing KP and KPL1 mice analyzed by BLI at week 16 after viral transduction. b , Growth curve of KP and KPL1 primary tumor burden as determined by BLI. KP, n = 37; KPL1, n = 14. ns, P > 0.9999. c , Micro-CT scan images of normal lung and tumor-bearing lungs from KP and KPL1 mice at week 22 after viral transduction. Arrows , tumors; red h , heart. d , Tumor burden comparison between KP and KPL1 mice based on micro-CT scans at week 22. n = 38 KP mice, n = 10 KPL1 mice. ns, P = 0.3192. e , Tumor areas of KP and KPL1 mouse lungs at week 22-30 after viral transduction. n = 4 mice. ns, P = 0.6857. f , Graphical representation of mouse survival prior to reaching the target tumor burden, set at a luciferase bioluminescence flux of 10 7 photons/sec per mouse chest. KP, n = 35; KPL1, n = 14. ns, P = 0.7143. g , Quantification of tumoroids formed per 10,000 cells over 7 days. KP, n = 6; KPL1, n = 6. ns, P = 0.6991. h , Schematic of three different metastasis inoculation routes. i , Representative image of ex vivo lung BLI signals at week 5 after intratracheal delivery of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. j , Quantification of ex vivo lung BLI signal in the experiment in panel ( i ). KP, n = 8; KPL1, n = 6. ** P = 0.0027. k , Representative image of ex vivo lung BLI signals at week 3 after intracardiac injection of single-cell suspension of KP and KPL1 tumoroids (5 x 10 4 cells) into athymic mice. l , Quantification of ex vivo lung BLI signals in the experiment in panel ( k ). KP, n = 9; KPL1, n = 5. ** P = 0.0070. m , Representative image of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). n , Quantification of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. o , Representative image of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). p , Quantification of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. q , Representative image of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). r , Quantification of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. s , Western immunoblotting analysis of Ru631 PDX cells transduced with two different L1CAM shRNAs. t , H&E staining of lung sections four weeks after injecting Ru631 L1CAM knockdown via tail vein. Scale bar, 50 μm. u , Quantification of Ru631 tumors after tail vein injection. n = 4 mice per condition. * P = 0.0337; ** P = 0.0013. v , Western immunoblotting of L1CAM knockdowns with two different short hairpins in Ru323 PDXs. w , H&E staining of lung sections four weeks after injecting Ru323 with L1CAM knockdown via tail vein. Scale bar, 50 μm. x , Quantification of Ru323 tumors after tail vein injection. n = 5 mice per condition. *P = 0.0123; ** P = 0.0058. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( d , e , j , l , n , p , r ). Error bar indicates mean ± S.D. ( b ). Statistical significance was assessed using Kolmogorov-Smirnov test ( b ), two-tailed Mann-Whitney test ( d , e , g , j , l , n , p , r ) or one-way analysis of variance followed by the Tukey test ( u , x ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Transduction, Micro-CT, Comparison, Luciferase, Ex Vivo, Suspension, Injection, Western Blot, Staining, Knockdown, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Schematic summary of lung developmental continuum from early to late progenitor stages defined by specific transcription factors. b , Representative 3D maximum intensity projections (MIP) images of SOX2, NKX2-1 and SOX9 IF in the anterior foregut domain in embryonic day E9.5 mouse embryos and the distal lung bud tip in E10.5 mouse embryos. The dashed box demarcates the SOX2/NKX2-1 boundary in E9.5 and NKX2-1/SOX9 boundary in E10.5 as illustrated in the zoomed 2D (Z-Slice) panels on the right. All data were independently validated from replicate samples of at least n = 4 embryos with similar results obtained. c , SOX2, NKX2-1, and SOX9 IF staining in patient-derived primary tumor and metastasis tissue sections. Magnified regions are highlighted in yellow boxes. Scale bar, 50 μm. d , Fraction of cancer cells expressing SOX2, NKX2-1, or SOX9 by IF analysis in patient-derived primary tumor and metastasis tissue sections. Mean ± S.D. n = 6 patient samples each. ns, P = 0.4848 (NKX2-1), 0.3095 (SOX9); ** P = 0.0043. e , SOX2 and SOX9 IHC staining in LUAD patient-derived primary tumor and pleural metastasis samples. Magnified regions are highlighted in red boxes. Scale bar, 100 μm. f , Box and whisker plots of SOX2 and SOX9 IHC H-score in the patient-derived primary tumor and metastasis samples. Primary, n = 15; Metastasis, n = 7. ns, P = 0.1229; ** P = 0.0011. g , Relative median survival of LUAD patients based on the expression of SOX2 , NKX2-1 , and SOX9 in the primary tumor. Gene expression and survival data compiled from GEO, EGA and TCGA. SOX2 (low, n = 706; high, n = 705); NKX2-1 (low, n = 1083; high, n = 1083); SOX9 (low, n = 1083; high, n = 1083). * P = 0.0219; *** P = 0.0003; **** P < 0.0001. h , H&E staining of lung tissue sections harboring metastatic colonies upon tail vein injection of KP tumoroid cells expressing control or Sox2 short hairpins (sh). Magnified regions are indicated in red boxes. Scale bar, 5 mm. i,j , Number per lung ( I ) and percent area ( j ) of metastases in the experiment of panel ( h ). n = 10. **** P < 0.0001. k , UMAP of scRNA-seq data from autochthonous KP tumors collected 32 weeks after viral instillation. n = 4,489 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and numbered. l , Heatmap showing imputed average LUAD developmental marker expression in the clusters from primary tumors ranked left to right according to the average L1cam expression level. m , UMAP of scRNA-seq data from KP tumoroids collected after 7 days. n = 12,962 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and are represented by a different color and a number. n , Heatmap showing imputed average LUAD developmental marker expression in the clusters from 7-day tumoroids ranked left to right according to the average L1cam expression level. Lung developmental progenitor stages represented by the predominant expression of Sox2 ( early developmental stage), Nkx2-1 and Foxa2 ( middle progenitor stage), Hmga2 and Sox9 ( late progenitor stage) are indicated. o , Representative L1CAM and SOX2 IF staining of the invasive front ( dotted yellow line ) within an autochthonous KP tumor (32 week post-Cre) and of KP-derived tumoroids grown for 4 days or 7 days. T , tumor; N , normal tissue. Magnified regions are indicated in red boxes. Scale bar, 50 μm for primary tumor; 20 μm for tumoroids. p , Relative Sox2 mRNA expression of KP tumoroid-derived cells sorted by L1CAM expression. Mean ± S.E.M. n = 3. * P = 0.0258. q , L1CAM and SOX2 IHC staining of representative LUAD PDXs with high or low expression of L1CAM. Scale bar, 50 μm. r , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD PDXs. Data are log-transformed for visualization. n = 36. s , L1CAM and SOX2 IHC staining in serial sections of patient-derived bone metastasis and liver metastasis tissues. Scale bar, 100 μm. t , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD patient primary and metastasis samples. Data are log-transformed for visualization. n = 61 samples (primary, 15; metastasis, 46). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( r,t ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f , i,j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( d , f , g , i , j ) or two-tailed t test after passing the Shapiro-Wilk normality test ( p ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Expressing, Immunohistochemistry, Whisker Assay, Gene Expression, Injection, Control, Marker, Transformation Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , SOX2 and NKX2-1 IF staining of patient-derived LUAD primary tumor tissue section. The invasive front is marked with a dotted yellow line, and the magnified low-grade and high-grade tumor areas are shown in red boxes. Scale bar, 1 mm. b , SOX2 and NKX2-1 IF staining of patient-derived primary tumor tissue section with different tumor grade areas. Invasive front is shown with a dotted yellow line. Scale bar, 20 μm. c , SOX2 and SOX9 IF staining in KP primary tumor and metastasis tissue sections collected simultaneously from the same mouse (35 week post-Cre). Scale bar, 100 μm. d , Western immunoblotting analysis of SOX2 expression after dox-induced Sox2 knockdown in KP tumoroids. e , f , Box and whisker plots of the imputed transcript levels of Sox2 ( e ) and L1cam ( f ) in the scRNA-seq transcriptional clusters from KP primary tumors. g , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from KP primary tumors. Data from transcriptional cluster 9 are shown in black. H , I , Box and whisker plots of the imputed transcript levels of Sox2 ( h ) and L1cam ( i ) from 7-day tumoroids. Data from the cluster 12 are shown in red. j , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from 7-day tumoroids. Data from transcriptional cluster 12 are shown in black. k , Proportion of SOX9 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein into athymic mice. KP, n = 10; KPL1, n = 5. ** P = 0.0013. l , Relative expression of SOX9 in two independent KP tumoroid lines upon conditional knockdown of L1CAM. Bar graphs, mean ± S.D. n = 3 in each condition. ns, P = 0.8301; * P = 0.0202, 0.0305; ** P = 0.0094. m , Western immunoblotting analysis of L1CAM levels after L1CAM overexpression in KPL1 tumoroids. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( e , f , h , i , k ). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( g , j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( k ) or one-way analysis of variance followed by the Tukey test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Western Blot, Expressing, Knockdown, Whisker Assay, Over Expression, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , SOX2 IF staining of KP and KPL1 primary tumors (32 week post-Cre). Scale bar, 10 μm. b , Percentage of SOX2 + cells in the experiment from panel ( a ). n = 3 mice for each condition. * P = 0.0411. c , d , Relative L1cam mRNA expression and Sox2 mRNA expression in KP and KPL1 tumoroids. KP, n = 4; KPL1, n = 4. * P = 0.0286 ( c , d ). e , IF images of KP and KPL1 tumors in the lung after injecting a high number (1 x 10 5 ) of single cells from KP and KPL1 tumoroids into athymic mice via the tail vein. Magnified regions are highlighted in yellow boxes. Scale bar, 100 μm. f , Proportion of SOX2 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein in athymic mice. KP, n = 10; KPL1, n = 5. * P = 0.0400. g , h , Relative expression of L1cam ( g ) and Sox2 ( h ) in two independent KP tumoroid lines upon Dox-dependent conditional knock down of L1cam . n = 4. **** P < 0.0001. i , j , Western immunoblot analysis ( i ) and quantification ( j ) of SOX2 and SOX9 levels in control and L1CAM-overexpressing (OE) KP tumoroids. n = 4. * P = 0.0286. k , l , H&E staining ( k ) and quantification ( l ) of subcutaneous tumors or lung metastases after injection of KPL1 cells with or without L1CAM overexpression into athymic mice, analyzed at 4 weeks after subcutaneous (subQ) injection or 5 weeks after tail vein (TV) injection. Scale bar, 1 mm (SubQ) and 100 μm (TV). n = 5. ns, P > 0.9999; * P = 0.0238. m , GFP (cancer cells) and SOX2 IF staining of control vs L1CAM overexpressing KPL1 cells in lung metastasis after 5 week-post tail vein injection. Magnified regions are highlighted in yellow boxes. Scale bar, 10 μm. n , Fraction of SOX2 + cells in the experiment from panel ( m ). Control, n = 5; L1CAM overexpression, n = 5. ** P = 0.0079. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f ). Bar graphs, mean ± S.D. ( c , d , g , h , j ) or ± S.E.M. ( b , l , n ). Statistical significance was assessed using the two-tailed t test after passing the Shapiro-Wilk normality test ( b ), two-tailed Mann-Whitney test ( c , d , f , j , l , n ) or one-way analysis of variance followed by the Tukey test ( g , h ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Expressing, Knockdown, Western Blot, Control, Injection, Over Expression, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Scatter plot of accumulated enrichment score and negative log-transformed false discovery rate (FDR) of pathways associated with the L1CAM + /SOX2 + tumoroid cell cluster defined by scRNA-seq. The pathway with the highest total enrichment score and the lowest FDR is labeled in red. b , Scatter plot showing the expanded list of individual WNT/PCP-related terms. c , Schematic representations of L1CAM and PCP complex components at a cell-cell junction. d , Heatmap displaying the average expression of core PCP components in the KP tumoroid cell clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. e , L1CAM and CELSR1 IF staining in KP tumoroid-derived cell monolayer. The merged image is magnified for visualization ( red box ). Scale bar, 10 μm. f , L1CAM and CELSR1 IF staining of lung metastasis one week after tail vein injection of H23 LUAD cells into athymic mice. Scale bar, 10 μm. g , Co-immunoprecipitation of L1CAM and the PCP component CELSR1 in H23 LUAD cell lysates. h , Quantification of L1CAM/CELSR1 PLA dots per cell in H23 LUAD cells. Negative PLA control, n = 25 cells; L1CAM/CELSR1 PLA, n = 31 cells. **** P < 0.0001. i , Control and L1CAM-knockout (KO) H23 cell monolayers were subjected to cytokeratin, CELSR1, and SOX2 IF staining. Scale bar, 20 μm. j , Fraction of SOX2 + cells quantified in control versus L1CAM-knockout H23 cell monolayers. Mean ± S.D. Control, n = 43 cells; L1CAM KO, n = 58 cells. * P = 0.0488. k , Western immunoblotting analysis of control and L1CAM-knockdown ( shL1CAM ) PDXs after incubating the cells with 1 μM bafilomycin A for 24 h. l , H&E staining of lung sections of athymic mice after tail-vein inoculation of L1CAM-knockout H23 cells with or without SOX2 overexpression. Scale bar, 20 μm. m , n , Box and whisker plots showing the number ( m ) and percent area ( n ) of metastatic lesions per lung in the experiments of panel ( l ). n = 5 for L1CAM KO-1; 10 for L1CAM KO-2. * P = 0.0317; ** P = 0.0012 in ( m ). ** P = 0.0079; *** P = 0.0002 ( n ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( h , m , n ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( h , j , m , n ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Transformation Assay, Labeling, Expressing, Staining, Derivative Assay, Injection, Immunoprecipitation, Control, Knock-Out, Western Blot, Knockdown, Over Expression, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , WNT/PCP pathway scoring as the top hit among highly enriched genes in L1CAM + /SOX2 + KP tumoroid cells. Top 3000 ranked differentially expressed genes in cluster #12 versus the rest of cell population in day-7 KP tumoroids were analyzed to determine uniquely enriched biological pathways from the Gene Ontology (GO), Reactome, and PANTHER databases. 30 pathways shared among these three analyses were concatenated into three related processes. 13 of these pathways were from the WNT/PCP-related process and had the lowest accumulated false discovery rate (FDR). Within the WNT/PCP-related processes, the PCP and non-canonical WNT signaling pathways showed the highest enrichment score. b , Heatmap displaying the average expression of core PCP components in the primary tumor clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. c , L1CAM and CELSR1 IF staining in KP tumoroids. The magnified region is indicated by red or white boxes . Scale bar, 10 μm. d , Image analysis workflow for detecting the colocalization of L1CAM and CELSR1 at cell-cell junctions using a steerable filter to extract curvilinear image features. e , Computer vision-driven detection and segmentation of L1CAM and CELSR1 at cell-cell junctions through steerable filtering. The thickness of segmented junctions was dilated for visualization purposes. Scale bar, 1 μm. f , L1CAM and CELSR1 PLA fluorescence in H23 LUAD cells. The PLA signals are shown as red dots with nuclei counterstaining. Scale bar, 10 μm. g , L1CAM western immunoblotting analysis in parental and L1CAM knockout H23 cells. h , Western immunoblotting analysis of SOX2 overexpression in H23 LUAD cells upon CRISPR/Cas9-mediated L1CAM knockout, clones 1 and 2. i. L1CAM and CELSR1 IF staining in HEK293T cells engineered to overexpress L1CAM versus control. Scale bar, 10 μm. j , L1CAM and CELSR1 segmentation analysis at cell-cell junctions in L1CAM overexpressing HEK293T cells. The region of higher magnification is indicated by a red box . k , Quantification of CELSR1 at cell-cell junctions in control or L1CAM overexpressing HEK293T cells. Control, n = 3406 junctions; L1CAM OE, n = 6508 junctions. **** P < 0.0001. l , L1CAM and FZD6 IF staining in control or L1CAM overexpressing HEK293T cells. Scale bar, 10 μm. m , Western immunoblotting analysis of SOX2 overexpression in CHD1 knockout H23 cells. n , Box and whisker plots showing the size of metastatic lesions per lung in SOX2 overexpressing, CHD1 knockout H23 cells. n = 9 for CHD1 KO-1; 10 for CHD1 KO-2. ns, P = 0.2805; * P = 0.0415. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( k , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( k ) or two-tailed Mann-Whitney test ( n ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Protein-Protein interactions, Expressing, Staining, Fluorescence, Western Blot, Knock-Out, Over Expression, CRISPR, Clone Assay, Control, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , FZD6 IF staining with counterstained nuclei in KP tumoroids upon conditional knockdown of L1cam . Scale bar, 10 μm. b , Relative intensity of FZD6 IF quantified in KP tumoroids upon conditional knockdown of L1cam . Left to right, n = 25, 23, 35, 29, 39, 41, 45, 53 tumoroids. * P = 0.0440, *** P = 0.0010. c , Relative Fzd6 mRNA level in upon conditional knockdown of Fzd6 in KP tumoroids. n = 4 experiments. **** P < 0.0001. d , Relative Sox2 mRNA level upon conditional knockdown of FZD6 in KP tumoroids. n = 4 experiments. ** P = 0.0040. e , IF staining for L1CAM, c-Jun(pS73), and SOX2 in KP tumoroids ( upper panels ) and image segmentation to quantify the signal ( bottom panels ). Scale bar, 10 μm. f , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and SOX2 in KP tumoroids. n = 649 cells. g , IF staining for cytokeratin, L1CAM, and c-Jun(pS73) in a patient-derived LUAD lymph node metastasis. Scale bar, 50 μm. h , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and L1CAM in patient-derived LUAD lymph node metastases. n = 2,166 cells from 12 different lymph nodes. i , c-Jun(pS73) IF staining and counterstained nuclei in KP tumoroids upon conditional knockdown of L1CAM. Scale bar, 20 μm. j , Percentage of cells staining positive for c-Jun(pS73) after conditional knockdown of L1cam in KP tumoroids. Left to right, n = 94, 67, 87, 101, 38, 41, 36, 36 tumoroids. **** P < 0.0001. k , Western immunoblotting analysis of L1CAM, S73 phosphorylated and total c-Jun levels in H23 LUAD cells treated with 10 μM (JNK-IN-8) JNK inhibitor (JNKi) for 2 h. l , Sox2 mRNA relative expression level upon incubation of KP tumoroids with 20 μM JNK inhibitor for 24 h. n = 7 for KP tumoroid #1; 4 for KP tumoroid #2. *** P < 0.0006; * P = 0.0286. m , Western immunoblotting analysis of S73 phosphorylated and total c-Jun levels upon the treatments with anisomycin for 6 h. n , Sox2 mRNA relative expression level upon incubation of KP cells with anisomycin for 6 h. n = 3. ** P = 0.0064 (left), 0.0021 (right). The bar graph indicates mean ± S.E.M. ( b - d , j , l , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( b - d , j , n ) or two-tailed Mann-Whitney test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Knockdown, Derivative Assay, Western Blot, Expressing, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Venn diagram of transcription factors and chromatin modifiers differentially active in L1CAM + /SOX2 + KP tumoroid cells (scRNA-seq cluster #12, ) based on CHEA and ENCODE databases. b , PLA image of CHD1 and c-Jun in H23 LUAD cells. The PLA signals are shown as red dots overlayed with nuclei staining. Scale bar, 10 μm. c , Quantification of PLA signals per nucleus. Control, n = 63 cells; CHD1/c-Jun, n = 39 cells. **** P < 0.0001. d , Co-immunoprecipitation of CHD1 and c-Jun in KP tumoroids. e , Metaplots of CHD1 only (green), c-Jun only (blue), and CHD1/c-Jun overlap (red) ChIP-seq peak summits relative to peak center of CHD1 (left) or c-Jun (right) in H23 cells. f , Venn diagram showing the overlap between CHD1 and c-Jun genome-wide peaks. g , ChIP-seq analysis of CHD1 and c-Jun binding to the Sox2 locus in H23 cells. Sox2 enhancers ( red ), promoter and gene body ( green ) are indicated. h , ChIP-qPCR analysis of CHD1 binding to the SOX2 promoter in H23 cells with and without JNK inhibitor. n = 3 experiments. *** P = 0.0002. i , Co-immunoprecipitation of CHD1 and the transcriptional elongation factor RTF1 in H23 LUAD cells. j , Western immunoblotting analysis of SOX2 levels upon CHD1 knockout in H23 LUAD cells. k , Western immunoblotting analysis of SOX2 levels upon CHD1 knockdown in KP tumoroids using two different shRNAs. l , H&E staining of lung sections from NSG mice after tail-vein inoculation of H23 cells with CRISPR/Cas9-induced knockouts of CHD1 or L1CAM. Magnified regions are shown ( red boxes ). Scale bar, 100 μm. m , n , Box and whisker plots showing the number ( m ) and percent area ( n ) of metastatic lesions per lung in the experiments of panel ( j ). n = 6 per experimental condition. ** P = 0.0035, 0.0060, 0.0015 from left to right; * P = 0.0301 in ( m ). ** P = 0.0026, 0.0028, 0.0011 from left to right; *** P = 0.0006 in ( n ). o , Model of L1CAM-dependent PCP activation of c-Jun/CHD1 driven SOX2 expression in LUAD progenitors to generate a metastasis-initiating state. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( c , m , n ). The bar graph indicates mean ± S.E.M. ( h ). Statistical significance was assessed using a one-way analysis of variance followed by the Tukey test ( h , m , n ) or two-tailed Mann-Whitney test ( c ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Control, Immunoprecipitation, ChIP-sequencing, Genome Wide, Binding Assay, ChIP-qPCR, Western Blot, Knock-Out, Knockdown, CRISPR, Whisker Assay, Activation Assay, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Cancer Therapeutics

Article Title: Nerve Growth Factor Links Oral Cancer Progression, Pain, and Cachexia

doi: 10.1158/1535-7163.mct-11-0123

Figure Lengend Snippet: Figure 2. Anti-NGF therapy decreased cancer proliferation in human cancer cells and mouse models. A, anti-NGF decreased cell proliferation rate of cultured HSC-3 cells at 24, 48, and 72 hours. B, anti-NGF treatment did not affect tumor size in the mouse paw model. All the tumor groups had significantly larger paw volumes compared with naive mice (P < 0.001). C, anti-NGF treatment greatly reduced tongue tumor size. D, Ki-67 immunointensity was decreased in a representative tongue tumor following anti-NGF treatment. E, quantification of Ki-67–positive cells in total 40,6-diamidino-2-phenylindole (DAPI)-positive cells showed significantly less Ki-67 activity following anti-NGF treatment. Horizontal scale bar, 100 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001. A, C, and E, 2-tailed Student's t test or Mann–Whitney U test was used; B, 2-way ANOVA multiple comparisons were used.

Article Snippet: In the mouse paw tumor model, anti-NGF antibody (Mab 256; R&D Systems; 12.5 mg in 20 mL PBS) or vehicle control (20 mLPBS)was injected into the right hind paw of mice starting on postinoculation day, 4 following the pain behavior measurement and twice a week thereafter until postinoculation day 21.

Techniques: Cell Culture, Activity Assay, MANN-WHITNEY

Journal: Molecular Cancer Therapeutics

Article Title: Nerve Growth Factor Links Oral Cancer Progression, Pain, and Cachexia

doi: 10.1158/1535-7163.mct-11-0123

Figure Lengend Snippet: Figure 3. Anti-NGF therapy decreased cancer-induced nociception. A, anti-NGF treatment greatly attenuated mechanical allodynia in cancer-bearing paws of mice, but had no effect on basal mechanical sensitivity. Two-way repeated-measures ANOVA showed a significant treatment effect (F ¼ 511.677, P < 0.001) and a time treatment interaction effect (F ¼ 1.661, P < 0.05); the anti-NGF group was significantly different from all other groups (P < 0.001). B, in the tongue cancer model, 2-way repeated-measures ANOVA showed a significant main effect of treatment (F ¼ 10.16, P < 0.001) with a significant treatment time interaction (F ¼ 2.29, P < 0.05). Mice treated with anti-NGF did not show the escalation in gnaw time but was shown by the control mice starting postinoculation day 21. C, immunohistochemistry of a representative trigeminal ganglion from an anti-NGF–treated tongue cancer mouse showed lower expression of TRPV1, TRPA1, and PAR-2 receptors. Scale bar, 100 mm. D, anti-NGF treatment markedly reduced TRPV1, TRPA1, and PAR-2 immunointensity in trigeminal ganglia of mice with tongue cancer. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: In the mouse paw tumor model, anti-NGF antibody (Mab 256; R&D Systems; 12.5 mg in 20 mL PBS) or vehicle control (20 mLPBS)was injected into the right hind paw of mice starting on postinoculation day, 4 following the pain behavior measurement and twice a week thereafter until postinoculation day 21.

Techniques: Control, Immunohistochemistry, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Nerve Growth Factor Links Oral Cancer Progression, Pain, and Cachexia

doi: 10.1158/1535-7163.mct-11-0123

Figure Lengend Snippet: Figure 4. Anti-NGF differentially modulates body weight in normal and cancer-bearing mice. A, 2-way repeated-measures ANOVA showed a significant main effect of treatment (F ¼ 10.16, P < 0.001) and time (F ¼ 7.63, P < 0.001). The anti-NGF–treated group was significantly different from tumor þ PBS and naive þ anti-NGF (P < 0.001), but was not different from tumor þ CL-anti-NGF group (P ¼ 0.09). Vehicle-controlled tumor mice had significant weight loss from their baseline at day 18 (a). In contrast, no weight loss is shown for anti-NGF–treated mice. Note that naive mice treated with anti-NGF had significant weight loss at day 18 (b). At days 18 and 21, tumor þ anti-NGF group had significantly larger body mass compared with tumor þ PBS or naive þ anti-NGF groups. B, in mice with tongue tumors, 2-way repeated-measures ANOVA showed a significant main effect of treatment (F ¼ 11.53, P < 0.001) with a significant treatment time interaction effect (F ¼ 2.8, P < 0.01). Anti-NGF treatment prevented weight loss starting at day 17. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05 tumor þ anti-NGF versus tumor þ PBS; ##, P < 0.01 tumor þ anti-NGF versus tumor þ PBS; þþ, P < 0.01 tumor þ anti-NGF versus naive þanti-NGF; þþþ, P < 0.001 tumor þ anti-NGF versus naive þ anti-NGF.

Article Snippet: In the mouse paw tumor model, anti-NGF antibody (Mab 256; R&D Systems; 12.5 mg in 20 mL PBS) or vehicle control (20 mLPBS)was injected into the right hind paw of mice starting on postinoculation day, 4 following the pain behavior measurement and twice a week thereafter until postinoculation day 21.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: Nerve Growth Factor Links Oral Cancer Progression, Pain, and Cachexia

doi: 10.1158/1535-7163.mct-11-0123

Figure Lengend Snippet: Figure 5. Anti-NGF in tongue cancer mouse models decreased plasma TNF-a (A) and IL-6 (B), and increased plasma and fat leptin (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control. Two-tailed Student's t test was used for data analysis.

Article Snippet: In the mouse paw tumor model, anti-NGF antibody (Mab 256; R&D Systems; 12.5 mg in 20 mL PBS) or vehicle control (20 mLPBS)was injected into the right hind paw of mice starting on postinoculation day, 4 following the pain behavior measurement and twice a week thereafter until postinoculation day 21.

Techniques: Clinical Proteomics, Control, Two Tailed Test

Journal: Molecular Cancer Therapeutics

Article Title: Nerve Growth Factor Links Oral Cancer Progression, Pain, and Cachexia

doi: 10.1158/1535-7163.mct-11-0123

Figure Lengend Snippet: Figure 6. Correlation of cytokines with cancer progression, nociception, and weight change in the tongue cancer model. A–C, plasma IL-6 is positively correlated with tumor size, gnaw time, and weight loss. D, TNF-a is positively correlated with weight loss. E and F, plasma and fat leptin are inversely correlated with weight loss. Open circles, control group; closed circles, anti-NGF–treated group. Simple linear regression was used to test correlation.

Article Snippet: In the mouse paw tumor model, anti-NGF antibody (Mab 256; R&D Systems; 12.5 mg in 20 mL PBS) or vehicle control (20 mLPBS)was injected into the right hind paw of mice starting on postinoculation day, 4 following the pain behavior measurement and twice a week thereafter until postinoculation day 21.

Techniques: Clinical Proteomics, Control

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is increased in liver metastases (Met) as compared to matched primary tumors (Pri). Immunohistochemistry (IHC) for L1CAM is shown in matched normal colon, primary CRC tumor and liver metastasis sections from a representative patient. Arrows indicate L1CAM staining at the invasion front of the primary tumor. Detail of the boxed region is shown in Extended Data Fig. 1a. b, The percentage of L1CAM-expressing tumor cells in each section. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 18 paired patient samples; two-sided Wilcoxon matched-pairs signed-rank test. c, Percentage of L1CAM-expressing cells in matched pretreatment (pre-treat.) biopsies and post-treatment (post-treat.) surgically resected residual disease in patients with locally advanced rectal cancer. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 31 patients; two-sided Wilcoxon matched-pairs signed-rank test. d, Representative sections of paired pretreatment core biopsies and matched surgical resection specimens obtained after chemoradiation, from two patients with rectal adenocarcinoma, showing L1CAM expression in peripheral areas of residual adenocarcinoma after treatment. e, L1CAM immunohistochemistry in a human CRC liver metastasis resected after neoadjuvant chemotherapy, showing dense stromal infiltration and L1CAM-expressing residual tumor cell clusters. Representative of 18 samples analyzed. f, Tumor L1CAM expression is associated with greater organoid generation capacity. Median L1CAM expression is shown for freshly resected and dissociated patient CRC liver metastases measured by flow cytometry before plating of 10,000 cells in 40 μl of Matrigel using organoid medium. Organoid generation ability was assessed 14 d after plating. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 14 paired patient tumor samples; two-sided Mann-Whitney U test. g, Number of organoids (mean ± s.e.m.) grown from 10,000 L1CAMhigh or L1CAMlow cells flow-sorted from freshly resected patient CRC primary tumors (P, left) or liver metastases (Li, right), counted 14 d after surgical resection and flow sorting. From left to right, n = 3, 3, 3, 3, 3, 3, 3, 3, 3, 4, 3, 4, 4 and 11 replicates per group from each of seven patients; two-tailed Student’s t tests. h, Subcutaneous tumor volumes measured 35 d after transplantation of mice with 50,000 organoid-derived flow-sorted L1CAMhigh or L1CAMlow cells (mean ± s.e.m.); n = 5 mice per group; two-tailed Mann-Whitney U test. i, Biaxial density plot showing relative expression of L1CAM and LGR5 in 9,974 cells from four independent patient-derived metastatic CRC organoids subjected to scRNA-seq. Five clusters identified according to relative L1CAM and LGR5 expression are overlaid as colors on the density plot. j, Dual LGR5 mRNA FISH and L1CAM immunofluorescence (IF) on a patient primary CRC tissue section (top, low magnification; bottom, high magnification), showing discrete expression levels of LGR5 and L1CAM in different cell clusters, including double-positive cells. Representative field of eight tumor sections from four patients analyzed.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Staining, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Transplantation Assay, Derivative Assay, Immunofluorescence

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM inhibition rescues the increase in organoid generation secondary to REST inhibition. MSK107Li organoids stably expressing the indicated shRNAs were grown in the presence or absence of doxycycline for 7 d before measuring cell viability (luminescence relative to day 0; in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 6 organoid cultures per group; two-sided Mann-Whitney U tests). b, Relative mRNA levels of L1CAM and REST on day 0, normalized to GAPDH, in organoid-derived cells transduced with lentiviruses directing expression of the indicated shRNAs in the presence or absence of doxycycline. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests. c, Left: schematic diagram showing how loss of epithelial integrity induces L1CAM expression during wound healing and tumor invasion, ultimately driving metastatic relapse. Right schematic diagram showing that loss of membrane E-cadherin in cells detached from their epithelial niche downregulates and displaces REST from the L1CAM enhancer, thus enabling L1CAM expression.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Inhibition, Stable Transfection, Expressing, MANN-WHITNEY, Derivative Assay, Transduction, Membrane

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative human CRC primary tumor invasion front showing an inverse relationship between L1CAM and Ki67 expression. Representative of 16 tumors analyzed. b, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative post-treatment human CRC liver metastasis demonstrating L1CAMhighKi67low cells in organized epithelial structures and L1CAMhighKi67high cells in disrupted epithelia. Representative of 16 tumors analyzed. c, Percentage of L1CAMhigh and L1CAMlow cells that are also Ki67high in regions of intact versus disrupted glandular epithelial architecture within CRCs. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 64, 54, 28 and 34 independent fields from 16 patient tumors; two-sided Mann-Whitney U tests. d, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of MSK107Li matched surgically resected patient CRC liver metastasis, metastasis-derived organoids and organoid-derived subcutaneous xenograft. Dashed red lines indicate the tumor-stromal boundary. Box plots indicate the percentage of Ki67+ cells among L1CAMhigh cells in the indicated sections. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 7, 9 and 9 independent fields; two-sided Mann-Whitney U tests. e,f, L1CAM is induced during normal epithelial organoid formation. Relative L1CAM mRNA levels (mean ± s.e.m.) are shown for human (e) and mouse (f) cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients or mice; two-sided Student’s t tests. g, Relative LGR5 mRNA levels (mean ± s.e.m.) in human cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients; two-sided Student’s t tests. h, L1CAM is induced during epithelial regeneration after colitis. C57BL/6J mice were given 3.5% DSS in their drinking water for 5 d, inducing maximal colitis by day 7, and were then maintained on water without DSS for 12 d. Mice were killed at each of the indicated time points, and their colons were collected, sectioned and either stained with Kreyberg-Jareg stain (blue, mucin; pink, collagen) or subjected to immunohistochemistry for L1CAM. Representative images of three independent experiments are shown. i, High-magnification view showing detail of L1CAM immunohistochemical staining throughout the length of the intestinal crypt, representative of three mice each from three independent experiments.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Expressing, MANN-WHITNEY, Derivative Assay, Isolation, Staining, Immunohistochemical staining

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM immunohistochemistry in a representative colon section from an L1CAMΔIEC mouse maintained on water with DSS, showing L1CAM staining restricted to submucosal neurons and no L1CAM expression in the epithelial cells of the crypt. Representative of three independent mice. b–f, L1CAM deficiency impairs epithelial healing following DSS-induced colitis. L1CAMfl/y and L1CAMΔIEC mice were given 3.5% DSS in their drinking water for 5 d, followed by 12 d of water without DSS before being killed. b, Kaplan-Meier survival curves. n = 26 mice per group from three independent experiments; two-sided Mantel-Cox test. c, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 26 mice per group; two-sided Mann-Whitney U test. d, Histological score (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. L1CAMfl/y, n = 24; L1CAMΔIEC, n = 12. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; two-sided Mann-Whitney U test. e, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in L1CAMΔIEC mice on day 14. Panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts of L1CAMΔIEC mice. Representative of three independent experiments. f–l, L1CAM deficiency in the progeny of LGR5-expressing cells impairs epithelial healing after DSS-induced colitis. f, Immunohistochemistry for GFP in a representative colon section from an L1CAMΔLGR5 mouse killed after 5 d of daily tamoxifen treatment. n = 3 mice. g, WTΔLGR5 and L1CAMΔLGR5 mice were treated as in f and killed, and their colon crypts were isolated and seeded for organoid generation. Representative flow cytometry assessment is shown of L1CAM expression 24 h after seeding. n = 2 mice per genotype. h, Schematic of experimental design. Mice were treated with tamoxifen for 5 d to induce Lgr5-GFP-IRES-creERT2 expression and subsequently treated with 3% DSS for 5 d, with maximal colitis by day 7. They were then maintained on water without DSS for a further 12 d before being killed. i, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in the distal colon in L1CAMΔLGR5 mice on day 14, while crypts are restored in L1CAMfl/y and WTΔLGR5 mice. The panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts in L1CAMΔLGR5 mice in comparison to L1CAMfl/y and WTΔLGR5 mice. Representative of 20 evaluable mice from two independent experiments. j, Kaplan-Meier plot showing cumulative survival of L1CAMfl/y, WTΔLGR5 and L1CAMΔLGR5 mice. n = 33 mice from two independent experiments; two-sided Mantel-Cox tests. k, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 33 mice from two independent experiments; two-sided Mann-Whitney U tests. l, Histological scores (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 20 evaluable mice from two independent experiments; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Staining, Expressing, Activity Assay, MANN-WHITNEY, Immunohistochemical staining, Immunostaining, Isolation, Flow Cytometry, Comparison

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM is required for organoid regeneration. CRC107Li organoid-derived cells were transduced with lentivirus directing the expression of either Cas9 alone or Cas9 with sgRNAs targeting L1CAM and allowed to grow under antibiotic selection for 14 d, when they were flow-sorted and seeded at a concentration of 2,000 cells per 40 μl of Matrigel in independent wells of a 96-well plate. The number of organoids (mean ± s.e.m.) established from each population 14 d after sorting and seeding is shown. From left to right, n = 10, 13 and 11 organoid cultures per group; two-tailed Mann-Whitney U test. b,c, L1CAM knockdown inhibits regrowth of multiple patient-derived organoids. Organoids derived from four patients with metastatic CRC were transduced with lentiviruses directing the expression of doxycycline (Dox)-inducible shRNA targeting L1CAM, expanded and, where indicated, treated with doxycycline for 48 h before dissociation and seeding at a concentration of 2,000 cells per 40 μl of Matrigel. Knockdown efficiencies of two independent L1CAM-targeting shRNAs in four patient-derived organoids (b) and relative cell viability on day 14 as compared to day 0 (mean ± s.e.m.) after plating of organoid-derived single cells (c) are shown. n = 6 organoid cultures per group; two-sided Student’s t tests. d, L1CAM is required for subcutaneous tumor growth in vivo. MSK107Li organoid-derived cells (50,000) expressing a doxycycline-inducible shRNA targeting L1CAM were injected subcutaneously into each flank of immunodeficient NSG mice. Where indicated, organoids were treated with doxycycline 2 d before transplantation and mice were maintained on a doxycycline diet for the duration of the experiment. Tumor volume (mean ± s.e.m.) was measured with calipers at the indicated time points after subcutaneous inoculation. n = 10 tumors from five mice per group; two-tailed Mann-Whitney U test. e, Representative image and quantification of tumor bioluminescence measured 35 d after inoculation. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 10 tumors from five mice per group; two-sided Mann-Whitney U test. f, Day 21 steady-state MSK107Li and MSK121Li organoids were incubated in medium containing 50 μM irinotecan, and L1CAM expression was measured in residual DAPI− cells 7 d later. Top- flow cytometry plots showing distribution of the data. Bottom: bars showing median fluorescence intensity of L1CAM expression in each population. From left to right, n = 6,512, 130, 8,542 and 49 cells per group, representative of three independent experiments. g, Single cells derived from CRC107Li organoids transduced with lentivirus directing expression of the indicated shRNAs were seeded at a concentration of 2,000 cells per 40 μl grown as organoids for 21 d and then treated with doxycycline and/or irinotecan (irino) as indicated. The viability assay shows the luminescence (mean ± s.e.m.) of each population relative to the luminescence at the time that drug treatment was started (day 0); n = 5 organoid cultures per group; two-sided Mann-Whitney U test. h, Solid-phase binding assay showing dose-response curves of recombinant human L1CAM-Fc binding to plates coated with equimolar concentrations of the indicated proteins. After washing, bound L1CAM-Fc was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG, HRP substrate was added and OD450 was measured. Data are shown as the mean ± s.e.m; n = 5 wells per time point, representative of three independent experiments; two-tailed Mann-Whitney U test. i, L1CAM mediates the interaction of dissociated CRC cells with laminin isoforms. Single cells derived from MSK121Li organoids (3,000) cultured in the presence or absence of doxycycline to knock down L1CAM were seeded in wells coated with 30 nM of the indicated proteins. After 1 h of adhesion and extensive washing, the percentage of adherent cells (mean ± s.e.m.) was measured as the relative luminescence of each well immediately after plating. n = 10 organoid cultures per condition; two-tailed Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Derivative Assay, Transduction, Expressing, Selection, Concentration Assay, Two Tailed Test, MANN-WHITNEY, shRNA, In Vivo, Injection, Transplantation Assay, Incubation, Flow Cytometry, Fluorescence, Viability Assay, Binding Assay, Recombinant, Cell Culture

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is dynamically regulated during organoid growth. L1CAM immunohistochemistry of CRC organoids of varying size shows progressive restriction of L1CAM expression to cells at the periphery and an overall decrease in L1CAM expression with increasing organoid size. Representative of six organoid lines analyzed. b, L1CAM is induced by dissociation of normal, primary tumor and metastatic human organoids. Relative L1CAM mRNA levels, normalized to GAPDH mRNA levels (mean ± s.e.m.), were measured in intact organoids versus organoid-derived single-cell suspensions plated in Matrigel and assayed 24 h after dissociation. n = 4 replicates per group; two-sided Student’s t tests. c, Time course of regenerating organoids derived from L1CAMhigh (left) and L1CAMlow (right) cells flow-sorted from day 21 MSK107Li (top) and MSK121Li (bottom) organoids. Histograms show the distribution of L1CAM expression (measured by APC fluorescence) in each population at the indicated time points after flow sorting. Representative of three independent experiments. d,e, Dynamic induction of the L1CAMhigh phenotype by a subset of pre-existing L1CAMlow cells. CRC107Li organoids were labeled with lentivirally expressed tdTomato or GFP, and flow-sorted tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow cells were mixed in equal proportions and allowed to regrow as organoids in the presence or absence of irinotecan. Flow plots of the distribution of tdTomato- and GFP-expressing cells (d) and the relative proportions of these cells in the population (e) are shown from monitoring by flow cytometry at the indicated time points after mixing; from left to right, n = 17, 107, 68, 251, 484 and 348 DAPI− single cells representative of three independent experiments with two organoid lines; two-sided chi-squared tests. f,g, Flow cytometry contour plots showing the distribution of L1CAM expression at the indicated time points in the presence or absence of chemotherapy (f) and median fluorescence intensity (MFI) of L1CAM expression (measured by APC) in cells derived from tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow precursors on each day (g), representative of three independent experiments with two organoid lines.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Fluorescence, Labeling, Flow Cytometry

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a,b, L1CAM is not required for intestinal adenoma formation. Male APCΔIEC and L1CAM/APCΔIEC mice were killed at 3 months of age, and their colons were collected, sectioned and examined for adenoma formation. a, Number of adenomas per mouse intestine. In box plots, boxes show the 25th–75th percentile with the median and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. b, Mean adenoma diameter per mouse. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. c–e, L1CAM inhibition impairs orthotopic rectal tumor engraftment. NSG mice were given 3% DSS in their water for 5 d and then maintained on water without DSS for 2 d before intraluminal transplantation with 2 × 105 cells from dissociated MSK107Li organoids expressing doxycycline-inducible shRNA targeting L1CAM or control shRNA. Where indicated, organoids were treated in vitro with doxycycline starting 2 d before transplantation, and mice were maintained on a doxycycline diet. Mice were killed 90 d after transplantation, and their colons were collected and examined for tumor engraftment. c, Percentage of mice with an engrafted orthotopic tumor. From left to right, the stacked bar graphs show n = 7, 7, 10, 13, 9 and 7 mice per group from three independent experiments; two-sided chi-squared tests. d,e, Tumor diameter per engrafted mouse (in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 6, 5, 6, 4, 5 and 1 mice from three independent experiments; two-sided Mann-Whitney U tests) (d) and representative H&E-stained sections (e). Arrows indicate tumour diameter. f,g, L1CAM inhibition impairs metastatic colonization of the liver. Cells (5 × 104) were derived from dissociated MSK107Li organoids with doxycycline-inducible expression of shRNA targeting L1CAM. Where indicated, organoids were treated with doxycycline starting 2 d before transplantation and mice were maintained on a doxycycline diet. Representative H&E-stained sections of liver metastases at the experimental endpoint (arrows indicate tumour diameter) (f) and quantification of ex vivo liver bioluminescence signal measured 60 d after transplantation (g) are shown. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 9, 10, 8, 9, 5 and 5 mice per group; two-sided Mann-Whitney U tests. h–l, L1CAM inhibition impairs local tumor expansion and metastasis from orthotopic cecal xenografts. h, Schematic of the experiment: cells (4 × 105) derived from MSK121Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected into the cecal submucosa. Mice were monitored until cecal tumors were evident by ex vivo bioluminescence imaging 3 weeks after injection, randomized on the basis of bioluminescence signal and maintained on or off a doxycycline diet for 7 weeks before being killed. i–l, Quantification of whole-mouse bioluminescence signal (i) and ex vivo bioluminescence signal in the cecum (j), liver (k) and lung (l), normalized to bioluminescence at the time of randomization. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 5, 6, 12 and 11 mice per group; two-sided Mann-Whitney U tests. m–o, Combination of L1CAM inhibition with chemotherapy impairs tumor growth to a greater extent than chemotherapy alone. m, Schematic of the experiment. Cells (2 × 105) derived from MSK107Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected subcutaneously; mice were randomized on the basis of bioluminescence intensity 5 weeks after injection and maintained on a doxycycline diet and/or treated weekly with irinotecan as indicated for 4 weeks before being killed. n,o, Ex vivo tumor volume (n) and mass (o), normalized to tumor bioluminescence at the time of randomization. From left to right, n = 10, 10, 8, 7, 10, 10, 10 and 9 tumors per group; mean ± s.e.m.; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: MANN-WHITNEY, Inhibition, Transplantation Assay, Expressing, shRNA, In Vitro, Staining, Derivative Assay, Ex Vivo, Transduction, Luciferase, Injection, Imaging

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, REST ChIP-seq analysis, showing diminution of the REST peak at the L1CAM intronic enhancer in dissociated organoid-derived cells, collected 16 h after dissociation, in comparison to intact organoids. Input control is also shown. LCA10 (gray) is not expressed in CRC organoids. Two independent organoid cultures from two patient-derived organoid lines were analyzed per condition. b, Relative mRNA levels (mean ± s.e.m.) of REST and L1CAM in intact MSK107Li organoids (day 0), cells collected 24 h after dissociation and plating as single cells (day 1), and cells collected at the indicated time points during organoid regeneration. Organoids were transduced with lentivirus constitutively expressing shRNA targeting REST or control shRNA. Gene expression was normalized to GAPDH mRNA levels. Day 1 shControl versus shREST.1: P < 0.0001 (REST), P < 0.0001 (L1CAM); day 1 shControl versus shREST.2: P = 0.007 (REST), P < 0.0001 (L1CAM); n = 4 organoid cultures per sample per time point; two-sided Student’s t tests. c, Relative expression of CDH1, REST and L1CAM (mean ± s.e.m.) in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. n = 4 organoid cultures per group; two-sided Student’s t tests. d, ChIP-PCR using antibodies against REST or isotype-control immunoglobulin in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. Fold enrichment (mean ± s.e.m.) is shown relative to the corresponding 2% input. PCR primers were selected to amplify immunoprecipitated DNA at the indicated positions relative to the L1CAM transcriptional start site. P values correspond to the comparison between shControl anti-REST and shCDH1 anti-REST; n = 3 organoid cultures per condition; two-sided Student’s t tests. e, Induction of L1CAM expression by E-cadherin knockdown can be rescued by REST but not by dominant-negative REST (dnREST). Relative mRNA levels of L1CAM, CDH1 and REST are shown in MSK107Li organoids stably expressing shRNA targeting CDH1 or control shRNA as well as cDNA expressing REST or dnREST. Gene expression was normalized to the mRNA levels of GAPDH. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: ChIP-sequencing, Derivative Assay, Comparison, Transduction, Expressing, shRNA, Immunoprecipitation, Dominant Negative Mutation, Stable Transfection

Journal: Frontiers in Pharmacology

Article Title: Jia-Wei-Kai-Xin-San, an Herbal Medicine Formula, Ameliorates Cognitive Deficits via Modulating Metabolism of Beta Amyloid Protein and Neurotrophic Factors in Hippocampus of Aβ 1-42 Induced Cognitive Deficit Mice

doi: 10.3389/fphar.2019.00258

Figure Lengend Snippet: Jia-Wei-Kai-Xin-San treatment induced transformation of neurotrophic factors precursors into mature forms in hippocampus of Aβ injection mice. (A) The treatment of JWKXS and huperzine A in the mice were same as that in . The hippocampus tissues were collected after 1-week treatment and the expressions of proNGF and NGF were analyzed by western blot analysis and ELISA kits. Afterward, the ratio of mNGF to proNGF was calculated. (B) Expressions of proBDNF and BDNF were determined and the ratio of BDNF to proBDNF was calculated as same as that of NGF. Comparisons between groups were carried out by a one-way ANOVA followed by a post hoc Bonferroni test. Values were expressed in the percentage of control group as Mean ± SEM ( n = 8). # p < 0.05 (compared with sham group); ∗ p < 0.05, ∗∗ p < 0.01 (compared with model group).

Article Snippet: Aβ amounts were determined by amyloid beta protein mouse ELISA Kit (Jinyibai Company, China). proNGF and NGF levels were determined by mouse proNGF ELISA kit and mouse NGF ELISA kit (CUSABIO, Houston, TX, United States). proBDNF and BDNF levels were determined by mouse proBDNF ELISA kit and mouse BDNF ELISA kit (Aviscera Bioscience, Santa Clara, CA, United States).

Techniques: Transformation Assay, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Control