Journal: Cancer research

Article Title: Cancer-associated fibroblasts promote aggressive gastric cancer phenotypes via heat shock factor 1-mediated secretion of extracellular vesicles

doi: 10.1158/0008-5472.CAN-20-2756

Figure Lengend Snippet: (A) Nude mice were injected subcutaneously with MC38 cancer cells alone, or co-injected with either recombinant THBS2 or Activin A followed by another injection of recombinant protein two days later. Tumor size measured by caliper is presented as mean ± SEM for N=8 mice per group (across 2 experiments). Repeated measures Two-way ANOVA using least-squares means to adjust for group pairwise comparisons was used for statistical analysis. (B-C) Western blot analysis of fractions obtained from Optiprep density gradient isolation of EVs secreted by WT MEFs blotted against exosomal markers ALIX and TSG101, as well as THBS1/2, INHBA and HSF1. EVs from 3 WT MEFs were pooled together for the isolation. The experiment was repeated twice (with different biological replicates), representative results are shown. (D) Representative transmission electron microscope (TEM) images of low (i-ii) and high (iii) density EV fractions (repeated 2 times, from 2 biological replicates). (i) – 1.03% sucrose; (ii) – 1.04% sucrose; (iii) – 1.07% sucrose. Scale bars- 100 nm. (E) Representative western blot showing INHBA levels from EVs isolated from the serum of tumor-bearing and naïve iLgr5;GLI2A mice. ALIX was used as loading control. Arrow indicates expected size of ALIX. (F) INHBA levels from EVs isolated from the serum of tumor-bearing and naïve iLgr5;GLI2A mice were analyzed using western blot. INHBA levels were normalized to ALIX. Average expression of INHBA normalized to ALIX in 5 biological replicates (across 2 experiments) is presented in as mean ± SEM. Two-tailed Student’s t-test was used for statistical analysis. (G-J) INHBA, THBS1 and THBS2 levels in EVs derived from WT and Hsf1 null primary MEFs were analyzed using western blot. ALIX and TSG101 were used as loading controls. Representative blots are shown in (G). (H) Average expression of INHBA normalized to TSG101 in 8 biological replicates (across 3 experiments for INHBA) is presented as mean ± SEM. (I) Average expression of THBS1 normalized to TSG101 in 5–7 biological replicates (across 3 experiments) is presented as mean ± SEM. (J) Average expression THBS2 normalized to TSG101 in 10–11 biological replicates (across 4 experiments) is presented as mean ± SEM. Two-tailed Student’s t-test was used for statistical analysis in (H-I). (K) Nude mice were injected subcutaneously with MC38 cancer cells alone, or co-injected with EVs derived from WT or Hsf1 null MEFs. Tumor size measured by caliper is presented as mean ± SEM for N=14–15 mice per group (across 4 experiments). Repeated measures Two-way ANOVA using least-squares means to adjust for group pairwise comparisons was used for statistical analysis. (L) Graphic summary of the proposed model. HSF1 in CAFs regulates expression of INHBA and THBS1/2. INHBA and THBS2 from CAFs are packaged into EVs and secreted to the TME, where they are taken up by cancer cells.

Article Snippet: Co-injection of recombinant Activin A and THBS2 with MC38 cancer cells into Nude mice MC38 (2*10 5 ) were incubated with either PBS, 2.5 μg of Activin A (#CYT −146, ProSpec), or 2.5 μg of THBS2 (#1635-T2, R&D Systems) and co-injected in a total volume of 100 μl subcutaneously into Nude mice (Harlan laboratories).

Techniques: Injection, Recombinant, Western Blot, Isolation, Transmission Assay, Microscopy, Control, Expressing, Two Tailed Test, Derivative Assay

Journal: Cancer research

Article Title: Cancer-associated fibroblasts promote aggressive gastric cancer phenotypes via heat shock factor 1-mediated secretion of extracellular vesicles

doi: 10.1158/0008-5472.CAN-20-2756

Figure Lengend Snippet: (A-E) INHBA, THBS1 and THBS2 protein expression levels in WT and Hsf1 null primary MEFs were analyzed by western blot. Representative blots are shown in (A-B). An arrow indicates the expected size of INHBA bands. (C) INHBA western blot results of 5–10 biological replicates (across 2 experiments) were quantified, normalized to actin and are presented as mean ± SEM. (D-E) THBS1 western blot results of 5 biological replicates (across 2 experiments) and THBS2 western blot results of 5–10 biological replicates (across 3 experiments) were quantified, normalized to actin and are presented as mean ± SEM. Two-tailed Student’s t-test was used for statistical analysis in (C-E). (F-K) WT and Hsf1 null MEFs were co-cultured with N87-GFP cells for 72h, and each cell type was grown in mono-culture as control. Co-cultures were sorted by flow cytometry using GFP. (F-H) The levels of the indicated genes in (GFP-negative) MEFs were determined by qPCR. Average expression in 6–8 biological replicates (across 3 experiments for INHBA and THBS1 and 2 experiments for THBS2), normalized to HPRT, ± SEM are presented. Two-way ANOVA was used for statistical analysis. (I) Representative GFP (upper panel), and brightfield (lower panel) images of mono and co-cultures are shown. N=3 biological replicates. Scale bar- 50 μm. (J) Representative FACS plots showing the percentage of N87-GFP cells co-cultured with WT (left) and Hsf1 null MEFs (right). N=3 biological replicates. (K) The average percentage (± SEM) of N87-GFP cells co-cultured with WT and Hsf1 null MEFs in 3 biological replicates is shown. Two-tailed Student’s t-test was used for statistical analysis. (L-N) HFF cells treated with siHSF1, siINHBA, siHSF1-INHBA-THBS2 (siCombined) or siControl as indicated were co-cultured with N87-GFP cells for 72h. The percentage of N87-GFP in the co-cultures averaged across 5–9 biological replicates (± SEM) (across 3 experiments for siINHBA and siHSF1-INHBA-THBS2 and 2 experiments for siHSF1) is shown. Two-tailed Student’s t-test was used for statistical analysis.

Article Snippet: Co-injection of recombinant Activin A and THBS2 with MC38 cancer cells into Nude mice MC38 (2*10 5 ) were incubated with either PBS, 2.5 μg of Activin A (#CYT −146, ProSpec), or 2.5 μg of THBS2 (#1635-T2, R&D Systems) and co-injected in a total volume of 100 μl subcutaneously into Nude mice (Harlan laboratories).

Techniques: Expressing, Western Blot, Two Tailed Test, Cell Culture, Control, Flow Cytometry

Journal: Cancer research

Article Title: Cancer-associated fibroblasts promote aggressive gastric cancer phenotypes via heat shock factor 1-mediated secretion of extracellular vesicles

doi: 10.1158/0008-5472.CAN-20-2756

Figure Lengend Snippet: (A-B) Gastric cancer was induced in iLgr5;GLI2A mice, PDPN+ fibroblasts were isolated from the resulting tumors and RNA-seq was performed using fibroblasts isolated from stomachs of naïve mice as control. Signatures comprised of genes upregulated (mCAF_up_sig;) or downregulated (mCAF_down_sig) in PDPN+ CAFs vs PDPN+ normal fibroblasts were derived. (A) KM analysis of overall survival in patients from the Singapore cohort stratified based on expression of the mCAF_up_sig (left) or mCAF_down_sig (right). (B) Enrichment of the mCAF_up_sig and mCAF_down_sig (mean of normalized counts) in patients with the MP and EP subtypes in the KUGH & KUCM cohort. One-way ANOVA was used for statistical analysis. (C) STRING analysis of potential interactions between protein products of genes differentially expressed in gastric cancer patients with favorable vs poor outcome. Proteins with no connections were omitted from the image. THBS2 and INHBA are highlighted in red. (D-F) Log normalized counts and p-adjusted values of the indicated genes taken from DESeq analysis of the iLgr5;GLI2A PDPN+ CAF RNA-seq data (Supplementary Table 8). (G-I) Total RNA levels of the indicated genes normalized to HPRT in normal stomachs and tumors (cancer) from iLgr5;GLI2A mice. N=3 mice per group, means ± SEM are presented. Two-tailed Student’s t-test was used for statistical analysis. (J-K) Representative images showing H&E and immunohistochemical staining of the indicated proteins in gastric tumors and control stomachs (naïve) from iLgr5;GLI2A mice. N=5 mice for cancer and N=3 mice for normal control. C- cancer, S- stroma. Scale bar- 100 μm. Arrows indicate INHBA and THBS1 positive CAFs.

Article Snippet: Co-injection of recombinant Activin A and THBS2 with MC38 cancer cells into Nude mice MC38 (2*10 5 ) were incubated with either PBS, 2.5 μg of Activin A (#CYT −146, ProSpec), or 2.5 μg of THBS2 (#1635-T2, R&D Systems) and co-injected in a total volume of 100 μl subcutaneously into Nude mice (Harlan laboratories).

Techniques: Isolation, RNA Sequencing, Control, Derivative Assay, Expressing, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Cellular immunology

Article Title: In vitro ovarian tumor-conditioned CD163+ human macrophages retain phagocytic response to CD47 blockade.

doi: 10.1016/j.cellimm.2025.104932

Figure Lengend Snippet: Fig. 2. Tumor conditioning induces macrophage CD163 and IL-10 gene expression. Tumor-conditioned human monocyte-derived macrophages (M(A2780)) were generated and their gene expression was compared to control macrophages by RT-qPCR. A-F: Presents gene expression of polarization markers (CD163, IL-10, and TNF-α) and phagocytosis checkpoints (SIRPα, LILRB1, and Siglec-10) in tumor-associated macrophages compared to controls. Statistics: comparisons were made against controls using paired t-tests and a significance level of p = 0.05. When indicated by a logarithmic y-axis, comparisons were made on log-transformed data. Data are presented as individual data points with mean ± SD. N = 4.

Article Snippet: The plates were read at 450/ 620 nm and a standard curve ranging from 0.625 to 8 μg/L was prepared using recombinant human LILRB1 (R&D systems, 8989-T2). sCD206 (samples diluted 1:5) [34], and sSIRPα (samples diluted 1:10) [35] were measured as previously reported in detail.

Techniques: Gene Expression, Derivative Assay, Generated, Control, Quantitative RT-PCR, Transformation Assay

Journal: Cellular immunology

Article Title: In vitro ovarian tumor-conditioned CD163+ human macrophages retain phagocytic response to CD47 blockade.

doi: 10.1016/j.cellimm.2025.104932

Figure Lengend Snippet: Fig. 3. Tumor cell conditioning induces CD163, CD206, CD80 and LILRB1 on the membrane protein level. Tumor-conditioned human monocyte-derived macrophages (M(A2780)) were generated and their membrane protein expression compared to control macrophages by flow cytometry. The gating strategy is presented in Supplementary fig. 1. A-G: Presents median fluorescence intensities (MFI) of polarization markers (CD163, CD206, and CD80) and MFI values and percent macrophages positive for phagocytosis checkpoints (LILRB1, and Siglec-10). Statistics: comparisons were made against controls using paired t-tests and a significance level of p = 0.05. When indicated by a logarithmic y-axis, comparisons were made on log-transformed data. Data are presented as individual data points with mean ± SD. N = 9.

Article Snippet: The plates were read at 450/ 620 nm and a standard curve ranging from 0.625 to 8 μg/L was prepared using recombinant human LILRB1 (R&D systems, 8989-T2). sCD206 (samples diluted 1:5) [34], and sSIRPα (samples diluted 1:10) [35] were measured as previously reported in detail.

Techniques: Membrane, Derivative Assay, Generated, Expressing, Control, Flow Cytometry, Fluorescence, Transformation Assay