Inhibition of integrin αVβ6 changes fibril thickness of stromal collagen in experimental carcinomas

Abstract

Background: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-β1 and -β3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma.

Methods: The potential role of α_Vβ₆ integrin-mediated activation of latent TGF-β was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal α_Vβ₆ integrin-specific monoclonal antibody 3G9 was used to inhibit the α_Vβ₆ integrin activity.

Results: Both KAT-4 and Capan-2 cells expressed the α_Vβ₆ integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-β in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-β1 and -β3 reduced collagen fibril thickness in both tumor models.

Conclusion: Our data demonstrate that the α_Vβ₆-directed activation of latent TGF-β plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to α_Vβ₆ inhibition depends on the simultaneous presence of alternative paths for latent TGF-β activation and the extent of desmoplasia.

Fig. 1

Luciferase-based assay showing TGF-β activity in mink lung epithelial cells (MLEC). a Effects by TGF-β elicited signaling in MLEC by heat-activated (CM heated) or non-activated (CM) conditioned medium from KAT-4 and Capan-2 cell cultures. b TGF-β activity in cultures of MLEC, KAT-4, Capan-2, and co-cultures of KAT-4 and MLEC (p = 0.0014). c Effect of 3G9 antibody on TGF-β activity in co-cultures of MLEC and KAT-4 cells. d Effect of Fc:TβRII on TGF-β activity in co-cultures of MLEC with KAT-4 cells. e Effect of 3G9 on TGF-β activity in KAT-4 and MLEC co-cultured with F99 fibroblasts and Capan-2 cells. f Effects of the ROCK inhibitor Y-27632 (10 μM) on TGF-β activity in fibroblasts co-cultured with tumor cells, as well as effects of TGF-β supplementation (10 nM). All data points were compiled from a minimum of three independent experiments performed in triplicates. Error bars are standard deviations. Asterisks indicate p < 0.05 as analyzed with Student’s t-test

Fig. 2

Comparison of gene expression in untreated KAT-4 (n = 3) and Capan-2 (n = 3) cells grown in three-dimensional collagen gels. mRNA expression (2log AU) of selected genes for ECM related transcripts in 3-dimensional collagen gels containing KAT-4 (black bars) and Capan2 (grey bars) Collagen α1(I) (Col1A1), collagen α2(I) (Col1A2), Matrix metalloprotease-13 (MMP-13), α2 smooth muscle actin (ACTA2), Platelet Derived Growth Factor A and B (PDGF-A and -B and PDGF receptor–α and -β (PDGFR –A and -B), procollagen-lysine, 2-oxoglutarate 5-dioxygenase − 1, − 2 and − 3 (PLOD -1, − 2 and − 3), Prolyl 4-hydroxylase subunit α-2 (P4HA2), Bone morphogenetic protein 1 (BMP-1), Lysyl oxidase (LOX), Hyaluronan Synthase − 2, and − 3 (HAS-2 and -3), Fibromodulin (FMOD), Decorin (DCN), Vimentin (VIM), Keratin-7,-17,-20 and − 80 (KRT-7,-17,-20 and − 80), Glucagone (GCG). Data was analyzed with Student’s t-test. Error bars are standard deviations

Fig. 3

Comparison of untreated KAT-4 and Capan-2 carcinoma models. a Hematoxylin and Eosin and Sirius red staining in KAT-4 and Capan-2 carcinomas (bars = 100 μm). Trichrome staining (bars = 20 μm) and immunofluorescence staining with biotinylated 3G9 antibody (green) shows that the expression of integrin α_Vβ₆ is located at the cell membrane in both KAT-4 and Capan-2 carcinomas (cell nuclei stained with DAPI, blue; bars = 50 μm). b Collagen content in untreated KAT-4 (n = 4) and Capan-2 (n = 5) carcinomas, represented by hydroxyproline mg/g wet weight. c Average growth of untreated KAT-4 (n = 8) and Capan-2 tumors (n = 7), represented in mm³ measured externally (length x width x height)

Fig. 4

Ultrastructural analysis of KAT-4 and Capan-2 tumors using transmission electron microscopy. a Collagen fibril diameter in Capan-2 tumors treated with Fc:TβRII (3 × 10 mg/kg) (n = 5). Mean diameter was 48 nm in PBS-treated (n = 5) and 39 nm in 3G9-treated tumors (p < 0.01). b Collagen fibril diameter in Capan-2 tumors treated with 3G9 (3 × 10 mg/kg) (n = 5). No significant differences (p > 0.5) were observed in collagen fibril diameter after 3G9 treatment (mean diameter was approximately 49 nm in both phenotypes). c Collagen fibril diameter in Capan-2 tumors treated with 3G9 (5 × 10 mg/kg, extended protocol). Mean fibril diameter was 47 nm in PBS-treated and 41 nm in 3G9-treated tumors (p < 0.02). d Collagen fibril diameter in KAT-4 carcinomas treated with 3G9 (2 × 3 mg/kg) (n = 3). Mean fibril diameter was 43 nm in PBS-treated and 38 nm in 3G9-treated tumors (p = 0.02) (n = 3). Inserts show representative micrographs of collagen fibrils. To analyze fibril distribution, each data point (diameter for each fibril) was tabulated in GraphPad using the Frequency distribution analysis tool. Data was analyzed with Student’s t-test

Fig. 5

a Interstitial fluid pressure (P_IF) in KAT-4 carcinomas treated with 3G9 at 3 (n = 8), 10 (n = 9), or 30 (n = 9) mg/kg, or with Fc:TβRII (10 mg/kg) (n = 9). PBS was used as negative control (n = 7). b Interstitial fluid pressure (P_IF) in Capan-2 tumors treated three times with 3G9 (n = 5) or with 3G9 long term (n = 6) at10 mg/kg. PBS was used as control (n = 11). c CD31-stained structures with an area > 29 μm² to avoid potential non-vascular CD31 positive structures in PBS- (n = 7) and 3G9-treated (n = 3) KAT-4 carcinomas. d CD31-stained structures measured as in C, but in PBS- (n = 4) and 3G9-treated (n = 5) Capan-2 carcinomas. Error bars are standard deviations