Figure 5 from Collagen-Bearing Exosomes from Breast Cancer–Associated Fibroblasts Promote T-cell Dysfunction

<p>Exosomal ECM from CAFs attenuates T-cell proliferation. <b>A,</b> Mean difference plot showing the log fold change and average abundance of each protein from CAF exosomes. Proteins with fold changes >1 are highlighted [red, up after proteinase K (PK) or blue, down after PK treatment]. Top 10 labeled. <b>B,</b> Gene set enrichment analysis identified proteins associated with ECM organization in CAF exosomes. <b>C,</b> Western blot of COL1A1 and COL5A2 proteins in cells (top) and in exosomes (bottom) of normal fibroblasts (NF) from the MFP of naïve animals and CAFs from the MCaP murine cell line. <b>D,</b> Western blot of COL1A1 and COL5A2 proteins in cells (top) and in exosomes (bottom) of untransformed normal human fibroblasts (NF) and CAFs from luminal breast cancer. <b>E</b> and <b>F,</b> Protein content based on BCA assay (<b>E</b>) and extracellular vesicle composition (<b>F</b>) of fractions 1–12 collected by iodixanol density gradient centrifugation. <b>G,</b> Western blot of fractions from ultracentrifugation, probed with antibodies against COL1A1, COL5A2, exosome markers Alix and CD63, and the serum protein albumin. <b>H,</b> Western blot of COL1A1 (top) and COL5A2 (bottom) in MCaP CAFs with empty vector (vector) or targeted sgRNA (sg) vectors. <b>I,</b> Exosomes from MCaP CAFs with empty vector or targeted sgRNA constructs were added to CFSE-labeled T cells along with anti-CD3/CD28 antibodies. CFSE dilution in T cells was measured by flow cytometry 72 hours later. <b>J,</b> Western blot of CAF exosomes after collagenase I digestion for 1 hour at indicated concentrations. <b>K,</b> CAF exosomes after digestion with collagenase I at indicated concentration were added to CFSE-labeled T cells along with anti-CD3/CD28 antibodies. CFSE dilution was measured by flow cytometry 72 hours later. Significance was tested using one-way ANOVA with Tukey honestly significant difference <i>post hoc</i> test (<b>I</b> and <b>K</b>). <b>L,</b> Naïve CD8⁺ T cells isolated from Balb/c mice were activated with anti-CD3/CD28 antibodies and nucleofected 72 hours later with Cas9 and sgRNAs targeting SHP2, DDR1, or LAIR-1, or with mock nucleofection (mock). Knockdown efficiency was assessed by Western blot. <b>M,</b> A subset of T cells from each cohort described in (<b>L</b>) was labeled with CFSE and subsequently stimulated with CAF-derived exosomes and anti-CD3/CD28 antibodies. Proliferation was assessed by flow cytometry 72 hours later. CFSE-labeled T cells stimulated with anti-CD3/CD28 antibodies alone served as positive controls (Stim.), whereas unstimulated cells (“Unstim.”) served as negative controls. Significance was tested using one-way ANOVA with Tukey honestly significant difference <i>post hoc</i> test.</p>