Journal: Alzheimer's & Dementia

Article Title: Assessing Aβ‐independent effects of Module 42 on immune function in vitro

doi: 10.1002/alz.71215

Figure Lengend Snippet: Assessing the effect of M42 recombinant proteins on intracellular Ca 2+ signaling in human induced pluripotent stem cell‐derived macrophages. (A) Summary graph showing the fold change in total Ca 2+ levels following the addition of recombinant M42 proteins. (B–G) Intracellular Ca 2+ levels following treatment with TMEFF2 ectodomain (B), MDK (C), SDC4 ectodomain (D), SFRP1 (E), SPON1 (F), and PTN (G). Left panels represent intracellular Ca 2+ levels defined as area under the curve (AUC). Right panels show representative traces from one biological repeat (n = 3 technical replicates). Intracellular Ca 2+ levels are measured as relative fluorescent units. Histamine (6 µM) was used as a positive control. N = 3 to 6 independent biological experiments (two independent macrophage factory set‐ups; four technical replicates/biological repeat). Error bars correspond to mean ± SD. Repeated measures one‐way ANOVA followed by Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.01.

Article Snippet: These include HTRA1 (2916‐SE‐020, Novus Biologicals; E. coli expression system), Fms‐related tyrosine kinase 1 (FLT1; FCL1058B, G&P Biosciences; mammalian expression system), SPON1 (3135‐SP‐025/CF, R&D Systems; mammalian expression system), GPC5 (2607‐G5‐050/CF, R&D Systems; mammalian expression system), and Olfactomedin‐like 3 (OLFML3; Acro Biosystems, OL3‐H52H4‐100ug; mammalian expression system).

Techniques: Recombinant, Derivative Assay, Positive Control

Journal: Alzheimer's & Dementia

Article Title: Assessing Aβ‐independent effects of Module 42 on immune function in vitro

doi: 10.1002/alz.71215

Figure Lengend Snippet: Assessing the effect of M42 protein treatment on viability of hiPSC derived macrophages. Human iPSC macrophages were treated with HTRA1 (A), DAG1 ectodomain (B), TMEFF2 ectodomain (C), MDK (D), GPC5 (E), SDC4 ectodomain (F), SFRP1 (G), SPON1 (H), and PTN (I) at 3 µg/mL, 1 µg/mL, and 100 ng/mL for 24 h. Viability was assessed and expressed as relative luminescence units (RLU). N = 2 or 3 independent biological experiments (one independent macrophage factory set‐up; three technical replicates/biological repeat). Error bars correspond to mean ± standard deviation (SD). Repeated measures one‐way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: These include HTRA1 (2916‐SE‐020, Novus Biologicals; E. coli expression system), Fms‐related tyrosine kinase 1 (FLT1; FCL1058B, G&P Biosciences; mammalian expression system), SPON1 (3135‐SP‐025/CF, R&D Systems; mammalian expression system), GPC5 (2607‐G5‐050/CF, R&D Systems; mammalian expression system), and Olfactomedin‐like 3 (OLFML3; Acro Biosystems, OL3‐H52H4‐100ug; mammalian expression system).

Techniques: Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Venn diagram outlining filtering for membrane and secreted genes that are enriched in the FP. The list was curated based on LC-MS identification of proteins from hFpO conditioned media, enriched expression in primary human FP cells from scRNA seq datasets, and presence in membrane/secreted GO terms. (B) Graph displaying ranked fold-difference in expression of membrane/secreted FP genes ordered from most conserved to most divergent expression. Heatmap represents the proportion of studies that found the plotted genes to be essential from the DepMap Portal. (C) Violin plots of gene expression for the most (>10-fold) divergently expressed membrane/secreted in humans (left) compared to mouse (right) primary FP cells. SPON1, NTN1, and NRP2 are included as conserved controls between species.

Article Snippet: The following primary antibodies were used: anti-NTN1 antibody (1:1000 dilution, rabbit, Abcam, ab126729), anti-SHH antibody (1:1000 dilution, rabbit, Abcam, ab53281), anti-GAPDH (1:3000 dilution, mouse, Cell Signaling Technology, 2118S), anti-GALNT2 (1:1000 dilution, rabbit, Invitrogen, PA5-21541), and anti-SPON1 (1:1000 dilution, goat, Bio-Techne, AF3135).

Techniques: Membrane, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Comparison of gene expression levels of 14,852 orthologous genes (gray points) between mouse (E9-14) and human (CS12-19) FP cells. Black dotted line indicates 1:1 gene expression while the blue dotted lines indicate 2-and 10-fold human enrichment. Colored points indicate membrane and secreted FP-enriched genes and indicate degree of gene expression divergence between human and mouse. (B) Schematic of workflow for generating hFpO from pooled KO hiPS cells and subsequently assembling with day 22 hSpO to form hMA in an arrayed format. At 3 d.a.f., the assembloids were cleared and confocal imaged to visualize commissural axons guiding towards the hFpO. (C) Heatmap displaying min/max normalized, fold-change in ROBO3 fluorescence intensity of hSpO axons 150 μm pre- and 150 μm post-crossing into the hFpO (n = 2-3 assembloids per gene). Two-way ANOVA performed on measurements the most distal 50 μm of heatmap; F 31,1972 = 10.05, P < 0.0001, following Dunnett’s multiple comparison test comparing Control 1 to all other conditions: ****P <0.0001 for Control 1 vs GALNT2 , ****P <0.0001 for Control 1 vs PLD3 , ****P < 0.0001 for Control 1 vs NTN1 , and **P = 0.0025 for Control 1 vs SPON1 . (D) Representative z-projected immunocytochemistry images of control and pooled KO hMA expressing ROBO3 and FOXA2 to visualize ROBO3+ axons projecting across the hFpO boundary. Scale bar, 100 μm. (E) Plot profiles of normalized fold-change ROBO3 fluorescence intensity of KO pool hFpO hMA 150 μm pre- and 150 μm post-crossing into the hFpO from (C). Dotted lines indicate the non-targeting control groups while the solid lines indicate the target gene group. Data is presented as mean ± SEM. Gray shaded region indicates the 50 μm bin where ROBO3 expression as a measure of axon guidance was assessed for statistical significance in (C). (F) Schematic outlining the method for generating isogenic KO hiPS cell lines from the axon guidance screen candidates and subsequent isogenic validation of phenotypes identified in the axon guidance screen. (G) Representative z-projected immunocytochemistry images of control and isogenic KO hMA expressing ROBO3 and FOXA2 at 2 d.a.f. Scale bar, 200 μm. (H) Bar plot displaying the proportion of ROBO3 coverage relative to the area of the isogenic KO hFpO candidates from 1-3 d.a.f (shades of grey; n = 4-5 assembloids per condition). Data is presented as mean ± SEM. Two-way ANOVA, F 4,59 = 121.8, P < 0.0001, following Tukey’s multiple comparison test ****P < 0.0001.

Article Snippet: The following primary antibodies were used: anti-NTN1 antibody (1:1000 dilution, rabbit, Abcam, ab126729), anti-SHH antibody (1:1000 dilution, rabbit, Abcam, ab53281), anti-GAPDH (1:3000 dilution, mouse, Cell Signaling Technology, 2118S), anti-GALNT2 (1:1000 dilution, rabbit, Invitrogen, PA5-21541), and anti-SPON1 (1:1000 dilution, goat, Bio-Techne, AF3135).

Techniques: Comparison, Expressing, Membrane, Fluorescence, Control, Immunocytochemistry

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Inference of CRISPR edits (ICE; no border) and KO-score (border) of CRISPR edited hFpO at day 8 (dark blue) and day 11 (light blue) of differentiation. (B) Gene expression analysis (by RT-qPCR) of FOXA2 in control and CRISPR edited hFpO at day 8 and 11 of differentiation (pooled mRNA from 3 individual organoids). Color scale indicates the relative expression of FOXA2 normalized to GAPDH . (C) Representative brightfield images of control and CRISPR edited hiPS cells (top) and z-projection of immunocytochemical images hFpO (stained for FOXA2 and Reddot2 nuclear stain) at day 8 and 11 of differentiation (bottom). Scale bar 100 μm. (D) Quantification of FOXA2/RedDot2 fluorescence intensity of control and CRISPR edited hFpO at day 8 (light gray) and 11 (dark gray) of differentiation (n = 2-3 organoids per condition). Data is normalized to negative control (hSpO). Data is presented as mean ± SEM. (E) ICE (no border) and KO-score (border) of isogenic CRISPR edited hiPS cell lines for GALNT2, NTN1 , and SPON1 knockout. (F) Western blot validation of hSpO and hFpO cell extracts and conditioned media collected from control and isogenic KO for GALNT2 (top), NTN1 (middle), and SPON1 (bottom). GAPDH immunoblotting was used as the sample loading control. (G) Gene expression analysis (by RT-qPCR) of FOXA2, PAX6 , and NKX2 . 2 in control and isogenic CRISPR edited hFpO at day 8 of differentiation. Each dot represents a separate differentiation experiment normalized to GAPDH expression. Data is presented as mean ± SEM. Two-tailed Mann-Whitney test was used. For FOXA2 : n = 4 control and 4 GALNT2 KO from 1 hiPS cell line; *P = 0.0286. For NKX2-2 : n = 4 control and 4 GALNT2 KO from 1 hiPS cell line; *P = 0.0286. For PAX6 : n = 4 control and 4 GALNT2 KO from 1 hiPS cell line; P = 0.0571.

Article Snippet: The following primary antibodies were used: anti-NTN1 antibody (1:1000 dilution, rabbit, Abcam, ab126729), anti-SHH antibody (1:1000 dilution, rabbit, Abcam, ab53281), anti-GAPDH (1:3000 dilution, mouse, Cell Signaling Technology, 2118S), anti-GALNT2 (1:1000 dilution, rabbit, Invitrogen, PA5-21541), and anti-SPON1 (1:1000 dilution, goat, Bio-Techne, AF3135).

Techniques: CRISPR, Expressing, Quantitative RT-PCR, Control, Staining, Fluorescence, Negative Control, Knock-Out, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: ( A ) Model showing the development of the KAL-LN2E1 metastatic LUSC cell line by in vivo passaging. ( B ) RNA-Seq data of genes that are differentially expressed in TIMs from both the LN2E1 and LN4K1 tumor models (across the 3 experimental conditions) when compared with their respective host strain BM-derived IMs (used as baselines). ( C ) Relative expression of top 10 genes with highest hazard ratios found to be overexpressed in TIMs by qPCR. ( D ) Spon1 expression in healthy versus tumor-bearing IMs from tumors and blood. ( E ) Plasma SPON1 concentrations (ng/mL) in healthy versus tumor bearing mice. ( F ) measure of SPON1 released from WT versus Spon1 –/– IMs. ( G ) SPON1 levels as seen in plasma taken from WT versus Spon1 –/– mice with orthotopic LLC tumors at day 19. ( H ) Total SPON1 expression for each cell type from human NSCLC single cell samples. **** P < 0.001, *** P < 0.001, ** P < 0.01, * P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: In Vivo, Passaging, RNA Sequencing, Derivative Assay, Expressing, Clinical Proteomics, Two Tailed Test

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: ( A ) Differentially expressed genes between SPON1 hi and SPON1 lo patients. GSEA following Fisher’s Exact Test. ( B ) Top 20 most highly significant gene sets enriched in the Hallmark, C5, and C2 data sets. ( C ) Specific enrichment plots for EMT (Hallmark), Collagen (C5), and TGF-β (Hallmark) pathways.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques:

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: ( A ) Schematic and representative LN2E1 spheroid formation assay in Matrigel with recombinant SPON1 treatment. ( B and C ) Spheroid area and numbers for LLC and spheroid area and numbers for LN2E1 with and without recombinant murine SPON1 treatment (5 μg/mL). ( D and E ) Collagen gene expression with SPON1 treatment in LLC spheroids and in LN2E1 spheroids. ( F ) Schematic of WT or Spon1 –/– IM coculture with LN2E1 or LLC spheroids. ( G ) Spheroid area and collagen gene expression with WT or Spon1 –/– IM coculture with LN2E1 spheroids. ( H ) Collagen gene expression with WT or Spon1 –/– IM coculture with LLC spheroids. ( I ) Collagen gene expression of LN2E1 and LLC spheroids with recombinant SPON1, WT IMs, Spon1 –/– IMs, or recombinant SPON1 plus Spon1 –/– IMs. **** P < 0.001, *** P < 0.001, ** P < 0.01, * P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons; 1-way ANOVA test for multiple comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Tube Formation Assay, Recombinant, Gene Expression, Two Tailed Test

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: ( A ) IVIS imaging at 15 days from injection of LLC tumors into either WT or Spon1 –/– mice. ( B ) Three-dimensional optical plus CT imaging at day 15 of LLC tumors in WT and Spon1 –/– mice with higher resolution quantification. ( C ) Weight and counts of tumor burden from LLC tumors in WT and Spon1 –/– mice. ( D ) Picrosirius red staining of FFPE tumors from WT and Spon1 –/– mice. ( E ) Quantification of collagen read outs for high-density matrix (HDM). ( F ) Schematic of CD8 depletion experiment. ( G ) IVIS results at day 13 of LLC tumors in WT or Spon1 –/– mice with Isotype or anti-CD8 antibody treatment. *** P < 0.001, ** P < 0.01, * P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons. Total original magnification, ×20.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Imaging, Injection, Staining, Two Tailed Test

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: ( A ) Schematic of adoptive transfer experiment with either WT or Spon1 –/– IMs infused into Spon1 –/– mice orthotopically injected with LLC cells. Infusions took place on days 6, 8, 11, and 12 after injections. ( B ) IVIS results on day 14 after injection. n = 3. ( C ) Gross histology visualization of disease burden. ( D ) Lymph node tumor weight and counts on day 14 after injection. n = 3. ( E ) Representative IVIS images of WT and Spon1 –/– mice with or without adoptive transfer of WT or KO IMs. ( F ) IVIS results on day 5 after injection. n = 7 each group. ( G ) IVIS results on day 13 after injection. n = 7 each group. ** P < 0.01, * P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons; 1-way ANOVA test for multiple comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Adoptive Transfer Assay, Injection, Two Tailed Test

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: ( A and B ) Collagen gene expression in both LN2E1 ( A ) and LLC ( B ) spheroids for negative control and LRP8 KO. ( C and D ) In vivo disease burden by tumor weight and counts in LN2E1 and LLC. ( E ) Collagen content in LLC shown by Sirius red high-density matrix levels. ( F ) In vivo IVIS results of LLC WT or LRP8-KO tumors in either WT mice or Spon1 –/– mice. n = 9 each group. ( G ) TGF-β1 as a top upstream regulator of the genes composing our Collagen Gene Signature. ( H and I ) Phospho-SMAD2 scoring of WT and Spon1 –/– LLC tumors and of negative control and LRP8-KO LLC tumors. Scale bars: 125 μM. ( J ) qPCR of Collagen and EMT genes of LN2E1 WT or LRP8-KO spheroids under treatment conditions of untreated, recombinant SPON1, TGF-βi (SB431542), recombinant SPON11 + TGF-βi, and KRFK peptide. **** P < 0.001, *** P < 0.001, ** P < 0.01, * P < 0.05. Data are shown as the mean ± SEM incorporating biological and technical replicate samples. Two-tailed Student’s t test for 2-group comparisons; 1-way ANOVA test for multiple comparisons.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Gene Expression, Negative Control, In Vivo, Recombinant, Two Tailed Test

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: ( A ) CCR2 + SPON1 + staining identifies SPON1 + TIMs in tumor cell islets (PanCK + ) in an LUAD tumor. PanCK, white; CCR2, red; SPON1, green; Hoechst (nuclear stain), blue. Scale bar: 25 μM. Arrowheads are pointing to a CCR2 + SPON1 + TIM. ( B ) Immunofluorescence staining of LUSC tumors showing cancer cells (aqua) expressing LRP8 (green) with TGF-β1 activation (+pSMAD2/red). Scale bars: 300 μM (full core), 50 μM (insets). ( C ) Sirius red staining of the same core used in B and an overlay with positive sirius red staining shown in white. ( D and E ) Two-sided Pearson correlations of percentage of pSMAD2 + LRP8 + panCK + cells with CCR2 + SPON1 + cell density ( P < 0.0001, r = 0.272) ( D ) and between pSMAD2 + LRP8 + panCK + cells and high-density matrix indices based on Sirius red staining ( E ) ( P = 0.008, r = 0.139). ( F ) Survival differences between patients expressing high and low HDM ( n = 164 patients, P = 0.0057).

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Staining, Immunofluorescence, Expressing, Activation Assay

Journal: JCI Insight

Article Title: Spon1 + inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling

doi: 10.1172/jci.insight.168792

Figure Lengend Snippet: Spon1 hi TIMs are recruited to the TME and bind to their receptor, LRP8, located on cancer cells. Through TGF-β1 activation, they enhance upregulation of our Collagen Gene Signature, which includes several fibrillar collagens. This then leads to extracellular collagen deposition and increased protumorigenic phenotypes such as migration, invasion, spheroid formation, and metastases.

Article Snippet: Either complete media or complete media with 5 μg/mL recombinant mouse SPON1 (R&D Systems, 7950-SP-050) was added after the matrix cell suspension solidified by placing at 37°C for 15–30 minutes.

Techniques: Activation Assay, Migration