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: Figure 1. Identifying Spon1 as an important mediator of LUSC disease progression. (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: Biomarker Discovery, 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: Figure 3. Spon1 increases fibrillar collagen and collagen remodeling gene expression in cancer cells. (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: Gene Expression, Tube Formation Assay, 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: Figure 4. Reduced tumor formation and collagen production in the absence of Spon1. (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: Figure 5. Phenotypic rescue of disease burden with restoration of Spon1+ TIMs. (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: Figure 6. Spon1 mediates its effects on disease progression and collagen content via LRP8. (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: Biomarker Discovery, 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: Figure 7. Patient NSCLC tumors show Spon1+ TIMs, and LRP8 expression on cancer cells and TGF-β1 signaling positively correlate with collagen expression, which leads to poor survival. (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+L- RP8+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: Expressing, Staining, Immunofluorescence, Activation Assay