Author response: Matrix-regulated integrin αvβ5 maintains α5β1-dependent desmoplastic traits prognostic of neoplastic recurrence (2016)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5β1-integrin. Mechanistically, we established that TGFβ is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvβ5-integrin redistribution of pFAK-independent active α5β1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5β1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool. https://doi.org/10.7554/eLife.20600.001 eLife digest Tumors are not entirely made out of cancerous cells. They contain many other components – referred to as tumor stroma – that may either encourage or hinder the tumor’s growth. Tumor stroma includes non-cancerous cells and a framework of fibrous sugary proteins, called the extracellular matrix, which surround and signal to cells while providing physical support. In the most common and aggressive form of pancreatic cancer, the stroma often makes up the majority of the tumor’s mass. Sometimes the stroma of these pancreatic tumors can protect the cancer cells from anti-cancer drugs. Researchers have therefore been interested in finding out exactly which aspects of the tumor stroma shield and support cancer cells, and which impede their growth and progression. Answering these questions could make it possible to develop new drugs that will change a tumor-supporting stroma into one that hinders the tumor’s growth and spread. The most abundant cells in the stroma of pancreatic tumors are called cancer-associated fibroblasts. Healthy specialized fibroblasts – known as pancreatic stellate cells – help to build and maintain the ‘normal’ extracellular matrix and so these cells normally restrict a tumor’s development. However, cancer cells can adapt healthy fibroblasts into cancer-associated fibroblasts, which produce an altered extracellular matrix that could allow the tumor to grow. Franco-Barraza et al. have now compared healthy and cancer-associated fibroblasts from patients’ pancreatic tumors. One of the main differences between these two cell types was the location of the activated form of a molecule called α5β1-integrin. Healthy fibroblasts, in a normal extracellular matrix, have active α5β1-integrin on the surface of the cell. However, a number of tumor-promoting signals, including some from the altered extracellular matrix, could force the active α5β1-integrins to relocate inside the fibroblasts instead. In further experiments, where the activated integrin was retained at the cell surface, the fibroblasts were able to resist the influence of the cancer-associated extracellular matrix. Then again, if the active α5β1-integrins were directed inside the cells, healthy cells turned into cancer-associated fibroblasts. With this information in hand, Franco-Barraza et al. examined tumor samples from over a hundred pancreatic cancer patients using a new microscopy-based technique that distinguishes cancer cells from stroma cells. The analysis confirmed the pattern observed in the laboratory: those patients who appeared to produce more normal extracellular matrix and have active α5β1-integrin localized mostly to the surface of the cells survived longer without the cancer returning than those patients who lacked these stroma traits. Samples from patients with kidney cancer also showed similar results and, as before, an altered extracellular matrix was linked to a worse outcome of the disease. Together these findings suggest that if future studies uncover ways to relocate or maintain active α5β1-integrin to the cell surface of fibroblasts they could lead to new treatments to restrict the growth of tumors in cancer patients. https://doi.org/10.7554/eLife.20600.002 Introduction The mesenchymal stroma typically found in normal tissues, which is composed mainly of naïve quiescent fibroblastic cells and their secreted interstitial extracellular matrix (ECM), constitutes a natural tumor suppressive microenvironment that enforces cellular homeostasis (Mintz and Illmensee, 1975; Soto and Sonnenschein, 2011; Xu et al., 2009; Bissell and Hines, 2011; Dolberg and Bissell, 1984; Petersen et al., 1992; Wong et al., 1992; Anderson et al., 2006; Wall et al., 2005). By contrast, the emergence of a desmoplastic (activated) stroma, encompassing fibrotic-like modifications of local and recruited fibroblasts based on tumor interactions with the local microenvironment, has been proposed to play a major role in the development and progression of solid tumors such as pancreatic ductal adenocarcinoma (PDAC) and others (Bijlsma and van Laarhoven, 2015; Jonasch et al., 2012; Erkan et al., 2007; Xu et al., 2015; Gupta et al., 2011). Many studies suggest that a fibrotic reaction, such as that seen in pancreatitis, drives a pro-tumorigenic wound-healing response that often precedes tumor development (Whitcomb and Pogue-Geile, 2002; Binkley et al., 2004). Tumor fibrosis, also known as desmoplasia, has been reported to promote tumorigenesis, providing chemoresistance and shielding tumors from therapeutic agents (Olive et al., 2009; Ireland et al., 2016; Laklai et al., 2016; Koay et al., 2014). A mechanism of ‘stromal reciprocation’, involving mutual signaling between tumor and neighboring cancer-associated fibroblasts (CAFs) that promotes tumor growth, has been demonstrated for PDAC (Tape et al., 2016). On the basis of these findings, several studies have attempted complete ablation of desmoplastic stroma as a therapeutic approach to limit tumor growth, but paradoxically, this resulted in the evolution of existing tumors to a more aggressive state, and accelerated rates of tumorigenesis (Özdemir et al., 2014; Rhim et al., 2014). By contrast, the idea of chronically ‘normalizing’ activated stroma by reprogramming desmoplasia from a tumor-promoting to a tumor-restrictive state has been suggested to bear greater therapeutic promise (Bijlsma and van Laarhoven, 2015; Sherman et al., 2014; Klemm and Joyce, 2015; Stromnes et al., 2014; Froeling et al., 2011), and the identification of a clinically applicable means to revert desmoplastic stroma is of considerable interest (Stromnes et al., 2014; Froeling et al., 2011; Alexander and Cukierman, 2016). Activation of local fibroblastic pancreatic stellate cells and recruited naïve fibroblastic cells during desmoplasia involves their transforming growth factor-beta (TGFβ)-dependent conversion to a myofibroblastic (activated) phenotype (Desmoulière et al., 1993; Meng et al., 2016; Principe et al., 2016; Xu et al., 2016). This phenotype is characterized by the induction of stress fibers and the elevated expression of alpha-smooth muscle actin (αSMA), palladin and other actin-binding proteins, and by the production of an aligned and organized (anisotropic) ECM, with parallel fibers that are rich in discrete fibronectin splice variants (e.g., ED-A) and in type I collagen (Mishra et al., 2007; Serini et al., 1998; Hinz, 2016; Rönty et al., 2006; Goetz et al., 2011). Despite the strong effect of TGFβ on fibroblast activation and ECM remodeling during epithelial cancer-associated desmoplasia, knowledge of the mechanisms and downstream consequences of this activation remain limited (Hesler et al., 2016; Oshima et al., 2015). In previous studies, we demonstrated that culturing naïve fibroblasts within CAF-secreted D-ECM is sufficient to induce myofibroblastic conversion (Amatangelo et al., 2005). Compatible with this dynamic ECM-dependent reprogramming, specific cell-matrix receptors, such as integrins αvβ5 and α5β1, have been identified as regulators of myofibroblastic αSMA (Asano et al., 2006; Lygoe et al., 2004) and as participants in the maturation of specific types of cell-matrix adhesions that support anisotropic fiber formation (Dugina et al., 2001). In the present work, we first asked whether D-ECM production and the ability of D-ECM to induce myofibroblastic activation can be decoupled and independently regulated. Using a patient-derived human pancreatic model (Lee et al., 2011) as main focus, with supporting data from human renal cancer (Gupta et al., 2011) and additional stromal (Amatangelo et al., 2005) models, we found that although TGFβ is needed for production of D-ECM, it is dispensable for subsequent D-ECM-induction of myofibroblastic activation of naïve fibroblasts. We also found that D-ECM controls αvβ5-integrin signaling, which prompts the accumulation of activated, but FAK-independent, α5β1-integrin pools in specific intracellular vesicles. This D-ECM control of αvβ5-integrin signaling prevents the enrichment of active α5β1-integrin at the plasma membrane (PM), where α5β1 activity opposes myofibroblastic activation. Using a novel integrative approach combining multi-colored immunofluorescence and a new quantitative algorithm, we first validated our in vitro findings and then applied this process to annotated clinical samples. This defined two readily distinguishable desmoplastic phenotypes that were correlated with markedly distinct clinical outcomes. These phenotypes are based on differences in the stromal localization and levels of either activated SMAD2/3 (indicative of TGFβ signaling) or active α5β1-integrin and FAK. These signatures help clarify the controversial role of desmoplasia in the progression of cancer. Further, insofar as reversion of D-ECM has been suggested have the potential to confer significant clinical benefit (Stromnes et al., 2014; Whatcott et al., 2015; Neuzillet et al., 2015), these data suggest potential therapies to stabilize patient-protective or to revert patient-detrimental stroma. Results TGFβ is necessary for CAF production of functional anisotropic D-ECM Fibroblasts were isolated from seven PDAC surgical specimens obtained from five different individuals (with four specimens reflecting two matched tumor-normal pairs, one tumor specimen lacking a matched normal control, and two specimens pathologically designated as non-tumor/normal). These fibroblasts were characterized as naïve pancreatic stellate cells or PDAC-associated desmoplastic CAFs on the basis of assessments of the mRNA and protein expression of the myofibroblastic markers palladin and αSMA (Figure 1A–B). All specimens were used in parallel for subsequent analyses. In primary culture, these fibroblasts produced characteristic ECM (Franco-Barraza et al., 2016). Desmoplastic CAFs produced anisotropic D-ECM with multi-layered myofibroblastic spindled nuclei and increased levels of stress fiber-localized αSMA reminiscent of myofibroblastic cells in vivo (Goetz et al., 2011; Provenzano et al., 2006; Conklin et al., 2011; Eyden, 2001; Kalluri and Zeisberg, 2006), whereas fibroblasts derived from normal specimens did not (Figure 1C). Quantification of ECM fiber alignment provided a robust measure of tumor-dependent fibroblast activation. We used an arbitrary quantitative threshold of at least 55% of fibers oriented at a spread of 15° from the mode angle as indicative of D-ECMs that had been produced by activated CAFs (Figure 1—figure supplement 1). Figure 1 with 3 supplements see all Download asset Open asset Characterization of human fibroblastic cells isolated from PDAC surgical samples. Fibroblastic cells were isolated from normal or tumoral surgical samples from PDAC patients. (A) Representative indirect immunofluorescent assessments of vimentin-positive and pan-cytokeratin-negative fibroblasts, isolated from PDAC surgical specimens. Harvested cells were probed for desmoplastic markers αSMA and palladin, while the pancreatic cancer cell line, Panc1, was used as an epithelial-to-mesenchymal transduced (EMT) control that is known to express both epithelial and mesenchymal markers. Assorted markers are shown in white while counterstained Hoechst-identified nuclei are shown in yellow. (B) The bar chart shows normal vs. desmoplastic mRNAs levels, corresponding to αSMA and palladin obtained by RT-qPCR from the indicated 3D-cultures following ECM production (obtained by confluent culturing of fibroblasts in the presence of ascorbic acid for a period lasting 8 days [Franco-Barraza et al., 2016]) (**p=0.0286). (C) Representative images of normal vs. desmoplastic phenotypes after 3D ECM production; comparison of low vs. high αSMA levels (white), heterogeneous/round vs. elongated/spindled nuclei (yellow) and disorganized/isotropic vs. parallel aligned/anisotropic ECMs (magenta) are evident in the representative images. Note that the examples shown corresponds to the matching pair of (naïve vs. desmoplastic) fibroblastic cells that were harvested from surgical samples corresponding to patient #1 and that this pair of cells was used for all examples provided in figures below, unless otherwise stated. https://doi.org/10.7554/eLife.20600.003 To first test if autocrine TGFβ signaling is essential for CAF production of TGFβ that then contributes to the formation of anisotropic D-ECM, we used an ELISA-based approach to measure levels of TGFβ present in D-ECM in comparison to levels found in normal ECM (N-ECM), or in ECM produced by CAFs treated with SB-431542, a small-molecule inhibitor of the TGFβ1 receptor. Levels of TGFβ in D-ECM were ~2 fold higher than in N-ECM or D-ECM produced in the presence of SB-431542 (D+TGFβi in Figure 1—figure supplement 2). Importantly, following SB-431542 treatment, CAFs produced isotropic ECMs that were phenotypically indistinguishable from intact N-ECMs (Figure 1—figure supplement 2B–D). This result differed from that produced by the treatment of control naïve pancreatic stellate cells with SB-431542, which produced interrupted TGFβ signaling that caused complete loss of fibrillogenesis, resulting in the absence of matrix production (Figure 1—figure supplement 3). Together, these results suggested that the increased TGFβ observed in D-ECM was critical for CAF production of anisotropic matrices. Stripping matrix-producing cells from their secreted ECMs produces a residual ‘extracted’ matrix into which new fibroblasts (e.g., naïve fibroblastic stellate cells) or cancer cells can be seeded (Franco-Barraza et al., 2016). Using primary human naïve fibroblastic cells, we have previously shown that the residual three-dimensional (3D) D-ECM produced by CAFs is sufficient to induce a myofibroblastic phenotype (Amatangelo et al., 2005). Applying this analysis to pancreatic extracted matrices, we found that D-ECM produced by CAFs in the presence of TGFβ blockade was similar to N-ECM in being incapable of inducing de novo myofibroblastic activation (as reflected by increased αSMA stress fiber localization and protein levels) in naïve fibroblastic stellate cells. By contrast,untreated and control treated D-ECM effectively induced such activation (Figure 2, Table 1). Similar results were obtained using D-ECM and naïve fibroblastic stellate cells for all five PDAC patients (Figure 2—figure supplement 1, and Table 2), suggesting that myofibroblastic activation is a general phenomenon during interactions between naïve pancreatic stellate cells and CAF-produced PDAC-associated D-ECMs. A post-translational effect was implied by the fact that αSMA mRNA levels were comparable in naïve fibroblastic stellate cells cultured in N- vs D-ECM (Figure 2—figure supplement 2A) and because use of cycloheximide to inhibit protein translation did not alter αSMA expression levels (Figure 2—figure supplement 2B), whereas αSMA localization differed (Figure 2). We also asked whether autocrine TGFβ signaling within naïve stellate cells is necessary for their myofibroblastic activation by D-ECM (Figure 2—figure supplement 3 and Table 3). Growth of these cells in the presence of SB-431542 did not block D-ECM-induced αSMA expression or localization to stress fibers. Table 1 αSMA stress fiber localization and expression levels in naïve fibroblasts (stellate cells) cultured overnight within assorted ECMs. https://doi.org/10.7554/eLife.20600.007 N-ECMD-ECMD+TGFβi D+DMSO αSMAStress fiber localization25% percentile0.170.820.320.85Median 0.58 1.00 0.55 1.02 75% percentile1.071.120.871.40expression25% percentile0.050.410.520.48Median 0.27 1.00 0.73 0.89 75% percentile0.781.501.141.19 Values obtained from naïve cells (pancreatic stellate cells isolated from patient #1) cultured overnight within intact D-ECMs (made from CAFs isolated from patient #1) were used for normalization and assigned an arbitrary unit of 1.00. Assorted, patient #1 derived, ECMs were intact N-ECM or intact D-ECM while experimental conditions included D-ECMs made by CAFs treated with SB-431542 (D+TGFβi) or DMSO (D+DMSO) during ECM production. Note that quantitative immunofluorescent obtained values of αSMA and F-actin were used to calculate stress fiber localization and expression of αSMA. P values, listed below, were calculated using the two-sided and two-tailed Mann Whitney test needed for normalized data. N-ECM vs. D-ECM; p<0.0001 stress fiber localization; p<0.0001 expression N-ECM vs. D+TGFβi; p=0.8847 stress fiber localization; p=0.0002 expression N-ECM vs. D+DMSO; p=0.0036 stress fiber localization; p<0.0001 expression D-ECM vs. D+TGFβi; p<0.0001 stress fiber localization; p=0.0198 expression D-ECM vs. D+DMSO; p=0.3578 stress fiber localization; p=0.1208 expression D+TGFβi vs. D+DMSO; p<0.0001 stress fiber localization; p=0.6236 expression Table 2 αSMA stress fiber localization and expression levels in assorted naïve pancreatic fibroblasts (stellate cells) cultured overnight within different D-ECMs. https://doi.org/10.7554/eLife.20600.008 (#X naïve cell) / (#Y D-ECM)αSMA Stress fiber localizationExpression 25% percentileMedian75% percentile25% percentileMedian75% percentile(2)/(1) 0.351.00 1.260.651.00 1.19(2)/(2) 0.901.15 1.380.871.10 1.30(3)/(2) 0.921.08 1.360.861.20 1.72(4)/(1) 0.080.85 1.030.792.62 3.04(4)/(5) 0.060.83 1.360.291.15 2.40(2)/(5) 0.521.24 1.330.601.44 2.31(2)/(1 +TGFβi) 0.000.00 0.040.040.07 0.10(2)/(5 +TGFβi) 0.000.06 1.200.010.14 0.76(3)/(1 +TGFβi) 0.010.06 0.230.010.15 0.28(4)/(5 +TGFβi) 0.110.22 0.710.250.74 1.66 Values obtained from naïve cells (e.g., inactive stellate cells) isolated from patient number ‘#2’ cultured overnight within D-ECMs made from CAFs isolated from patient #1 were used for normalization and assigned an arbitrary unit of 1.00. Assorted, naïve cells (patient numbers indicated) were cultured within D-ECMs derived from the indicated CAFs, while experimental conditions included assorted D-ECMs treated with SB-431542 (D+TGFβi) during ECM production. Note that quantitative immunofluorescent obtained values of αSMA and F-actin were used to calculate stress fiber localization and expression of αSMA. P values, listed below, were all calculated using the Mann Whitney test, compared to the normalized (2)/(1) experimental condition, to account for non-paired, two-tailed and non-Gaussian distributions of the data. (2)/(1) vs. (2)/(2); p=0.0836 stress fiber localization; p=0.3825 expression (2)/(1) vs. (3)/(2); p=0.1680 stress fiber localization; p=0.1736 expression (2)/(1) vs. (4)/(1); p=0.4266 stress fiber localization; p=0.0755 expression (2)/(1) vs. (4)/(5); p=0.8927 stress fiber localization; p=0.7509 expression (2)/(1) vs. (2)/(5); p=0.1885 stress fiber localization; p=0.4192 expression (2)/(1) vs. (2)/(1+TGFβi); p<0.0001 stress fiber localization; p<0.0001 expression (2)/(1) vs. (2)/(5+TGFβi); p=0.0909 stress fiber localization; p=0.0040 expression (2)/(1) vs. (3)/(1+TGFβi); p=0.0018 stress fiber localization; p<0.0001 expression (2)/(1) vs. (4)/(5+TGFβi); p=0.1018 stress fiber localization; p=0.5181 expression Table 3 αSMA stress fiber localization and expression levels in naïve fibroblasts (stellate cells) cultured overnight in the presence or absence of TGFβ inhibitor within intact D-ECMs. https://doi.org/10.7554/eLife.20600.009 TGFβ-i DMSO αSMA Stress fiber localization 25% percentile0.710.72Median 0.96 1.00 75% percentile1.291.04expression 25% percentile0.440.49Median 0.62 0.94 75% percentile1.301.41 Values obtained from naïve cells cultured overnight within intact D-ECMs were used for normalization (as shown in Table 1) and assigned an arbitrary unit of 1.00. TGFβ-i is the experimental condition in which naïve pancreatic stellate cells were cultured overnight in the presence of SB-431542 within intact D-ECM. DMSO treatment corresponds to vehicle control. Note that quantitative immunofluorescent obtained values of αSMA and F-actin were used to calculate stress fiber localization and expression of αSMA. P values, listed below, were calculated using the two-sided and two-tailed Mann Whitney test needed for normalized data. TGFβi vs. DMSO; p=0.3508 stress fiber localization; p=0.3361 expression TGFβi vs. N-ECM (from Table 1); p=0.0110 stress fiber localization; p=0.0010 expression TGFβi vs. D-ECM (from Table 1); p=0.9132 stress fiber localization; p=0.3401 expression DMSO vs. N-ECM (from Table 1); p=0.0036 stress fiber localization; p<0.0001 expression DMSO vs. D-ECM (from Table 1); p=0.2635 stress fiber localization; p=0.7408 expression Figure 2 with 3 supplements see all Download asset Open asset TGFβ is necessary for functional CAF-produced D-ECM. Naïve PDAC fibroblasts were cultured overnight within normal (N-ECM) vs. desmoplastic (D-ECM) ECMs that were produced in the presence or absence of TGFβ1-receptor inhibitor (D + TGFβi) or vehicle control (D + DMSO). All samples were subjected to indirect immunofluorescent labeling of αSMA and counterstained with fluorescently labeled phalloidin to detect actin stress fibers (F-actin). (A) Monochromatic images indicating double-labeled staining for αSMA and F-actin. (B) Quantification of the levels of localization of αSMA to actin stress fibers (F-actin) from (A). (C) Pseudo-colored images representing intensity-maps of αSMA levels, with an intensity color bar scale (0–255 intensity tone values) shown to the right. (D) Quantification of αSMA intensity from (C). Untreated D-ECM conditions were included in all in this and as normalization controls arbitrary conditions that induce myofibroblastic activation conditions that did not induce myofibroblastic activation. All and can be found in Table Together, these results suggested that TGFβ during D-ECM production the ability of CAFs to produce ECM that can induce myofibroblastic activation, but that D-ECM has been by CAFs, TGFβ retained within the D-ECM is dispensable for D-ECM-induced myofibroblastic activation of naïve stellate cells. D-ECM controls αvβ5 signaling to active α5β1-integrin during activation αvβ5 and α5β1 have been reported to in myofibroblastic activation (Asano et al., 2006; Lygoe et al., et al., 2001). We first established that both of these integrin were compared to other on the of human naïve pancreatic stellate cells and of PDAC-associated CAFs (Figure To whether either or both of these is essential for D-ECM induction of myofibroblastic naïve fibroblastic stellate cells were overnight on N-ECM or D-ECM in the presence of or control a specific et al., the ability of D-ECM to induce αSMA stress fiber localization levels induced by N-ECM (Figure and Table By contrast, which human α5β1-integrin activity et al., had limited on D-ECM induction of αSMA localization with naïve cells robust myofibroblastic conversion (Figure and Table application of and the of αvβ5-integrin seen with with naïve cells robust myofibroblastic similar to that in and controls (Figure and Table This the that of α5β1 downstream of of αvβ5 in D-ECM Table αSMA stress fiber localization and expression levels in naïve fibroblasts (stellate cells) cultured overnight within D-ECMs in the presence of integrin functional αSMA Stress fiber localization 25% 1.00 75% 25% 75% Values obtained from naïve cells cultured overnight within intact D-ECMs treated with or control Values obtained in D-ECMs (from Table 1) were used for normalization and assigned an arbitrary unit of 1.00. Note that the quantitative values of αSMA and F-actin obtained by immunofluorescence were used to calculate stress fiber localization and expression of αSMA. P values, listed below, were calculated using the two-sided and two-tailed Mann Whitney test needed for normalized data. vs. stress fiber localization; p=0.0002 expression vs. stress fiber localization; expression vs. stress fiber localization; expression vs. stress fiber localization; expression Figure 3 with 2 supplements see all Download asset Open asset αvβ5 α5β1 activity D-ECM-induced activation. (A) cell test was used to the expression of integrin in primary fibroblasts isolated from normal vs. matched tumor Note that differences were between the two cell types with to levels of αvβ5 and α5β1 (B) Naïve human pancreatic fibroblastic stellate cells were D-ECMs overnight in the presence of functional et al., functional et al., of both functional + functional et al., or Representative images of and F-actin fibroblasts are (C) Quantification of the in (D) images the intensity of αSMA expression including a color bar scale (0–255 intensity tone Quantification of Note that corresponding and for results shown in are in Table Naïve fibroblasts were D-ECMs produced by cell CAFs (Amatangelo et al., and subjected to αvβ5-integrin and α5β1-integrin et al., and or in The on myofibroblastic activation were for αSMA as in The the in in the for the intact control. The is shown for the experimental conditions in the additional a of the of cells myofibroblastic the of cells that have a stress fiber localized phenotype is shown Note that of α5β1-integrin effectively the phenotype that was by of as seen for the human PDAC conditions that resulted in myofibroblastic activation, while conditions that did not result in myofibroblastic activation. of D-ECM-induced activation of naïve fibroblasts, on the activity of integrins αvβ5 and of integrin results in of