(A) An integrin-dependent cell adhesion array test was used to assess the PM expression of integrin heterodimers in primary fibroblasts isolated from normal (white bars) vs. matched tumor tissue (desmoplastic; dark bars). Note that no differences were apparent between the two cell types with regards to levels of αvβ5 and α5β1 integrins. (B) Naïve human pancreatic fibroblastic stellate cells were re-plated onto D-ECMs overnight in the presence of functional blocking anti-αvβ5-integrin (ALULA [Su et al., 2007]; αvβ5-i), functional blocking anti-α5β1-integrin (mAb16 [Akiyama et al., 1989]; α5β1-i), combinations of both functional blocking antibodies (β5-i + α5-i), functional stabilizing anti-α5β1-integrin (SNAKA51 [Clark et al., 2005]; α5β1-act), or non-immunized isotypic antibodies (IgG). Representative monochromatic images of αSMA- and F-actin stained fibroblasts are shown. (C) Quantification of the experiment performed in (B). (D) Pseudocolored images depicting the intensity of αSMA expression including a color bar scale (0–255 intensity tone values). (E) Quantification of (D). Note that corresponding quantifications and p-values, for results shown in (B–E) are summarized in Table 4. (F) Naïve murine skin fibroblasts were re-plated onto murine D-ECMs (mD-ECM) produced by murine skin squamous cell carcinoma associated CAFs (Amatangelo et al., 2005), and subjected to αvβ5-integrin and α5β1-integrin inhibitors alone (ALULA: αvβ5-i [Su et al., 2007] and BMA5: α5β1i) or in combination (β5+ α5-i). The effects on myofibroblastic activation were measured for αSMA as in (B). The red asterisk illustrates the area outlined in red in the magnified insert for the intact (untreated) control. The same magnification is shown for the experimental conditions in the additional panels. As a method of quantifying the percentage of cells showing myofibroblastic features, the percentage of cells that have a stress fiber localized (αSMA) phenotype is shown (****p<0.0001). Note that inhibition of α5β1-integrin effectively reinstituted the mD-ECM-induced phenotype that was lost by inhibition of αvβ5-integrin, just as seen above for the human PDAC system. Checkmarks identify conditions that resulted in myofibroblastic activation, while Xs identify conditions that did not result in myofibroblastic activation. (G) Model of D-ECM-induced activation of naïve fibroblasts, dependent on the activity of integrins αvβ5 and α5β1. Inhibition of αvβ5- integrin results in release of active α5β1-integrin, leading to blockade of D-ECM-induced myofibroblastic activation (1st arrow, red X). The activity of αvβ5- integrin is no longer needed in the absence of α5β1-integrin activity, suggesting that α5β1-integrin activity in not necessary for fibroblasts to undergo D-ECM-induced myofibroblastic activation (2nd arrow, green checkmark). Double inhibition of αvβ5-integrin and α5β1-integrin results in D-ECM myofibroblastic activation, which proposes that inhibition of α5β1-integrin can overcome or rescue the effects seen under αvβ5-integrin inhibition (3rd arrow, green checkmark). Stabilization of α5β1-integrin in its active conformation overcomes the inhibitory/regulatory effects imparted by αvβ5-integrin, resulting in ineffective D-ECM-induced myofibroblastic activation (4th arrow, red X). Overall, the model suggests that D-ECM induces αvβ5- integrin activity, which in turn results in the regulation of active α5β1-integrin, allowing D-ECM-induced myofibroblastic activation (large arrow to the right, green checkmark).