Kristofer Rubin

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. 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. 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. 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.

New Findings: What is the central question of this study? Collagen‐binding β₁‐integrins function physiologically in cellular control of dermal interstitial fluid pressure (P_IF) in vivo and thereby participate in control of extravascular fluid volume. During anaphylaxis, simulated by injection of compound 48/80, integrin α_Vβ₃ takes over this physiological function. Here we addressed the question whether integrin α_Vβ₃ can replace collagen‐binding β₁‐integrin to maintain a long‐term homeostatic P_IF. What is the main finding and its importance? Mice lacking the collagen‐binding integrin α₁₁β₁ show a complex dermal phenotype with regard to the interstitial physiology apparent in the control of P_IF. Notably dermal P_IF is not lowered with compound 48/80 in these animals. Our present data imply that integrin α_Vβ₃ is the likely candidate that has taken over the role of collagen‐binding β₁‐integrins for maintaining a steady‐state homeostatic P_IF. A better understanding of molecular processes involved in control of P_IF is instrumental for establishing novel treatment regimens for control of oedema formation in anaphylaxis and septic shock. Accumulated data indicate that cell‐mediated contraction of reconstituted collagenous gels in vitro can serve as a model for cell‐mediated control of interstitial fluid pressure (P_IF) in vivo. A central role for collagen‐binding β₁‐integrins in both processes has been established. Furthermore, integrin α_Vβ₃ takes over the role of collagen‐binding β₁‐integrins in mediating contraction after perturbations of collagen‐binding β₁‐integrins in vitro. Integrin α_Vβ₃ is also instrumental for normalization of dermal P_IF that has been lowered due to mast cell degranulation with compound 48/80 (C48/80) in vivo. Here we demonstrate a role of integrin α_Vβ₃ in maintaining a long term homeostatic dermal P_IF in mice lacking the collagen‐binding integrin α₁₁β₁ (α11^−/− mice). Measurements of P_IF were performed after circulatory arrest. Furthermore, cell‐mediated integrin α_Vβ₃‐directed contraction of collagenous gels in vitro depends on free access to a collagen site known to bind several extracellular matrix (ECM) proteins that form substrates for α_Vβ₃‐directed cell attachment, such as fibronectin and fibrin. A streptococcal collagen‐binding protein, CNE, specifically binds to and blocks this site on the collagen triple helix. Here we show that whereas CNE perturbed α_Vβ₃‐directed and platelet‐derived growth factor BB‐induced normalization of dermal P_IF after C48/80, it did not affect α_Vβ₃‐dependent maintenance of a homeostatic dermal P_IF. These data imply that dynamic modification of the ECM structure is needed during acute patho‐physiological modulations of P_IF but not for long‐term maintenance of a homeostatic P_IF. Our data thus show that collagen‐binding β₁‐integrins, integrin α_Vβ₃ and ECM structure are potential targets for novel therapy aimed at modulating oedema formation and hypovolemic shock during anaphylaxis.

Cell-mediated contraction of collagenous matrices is modulated by various growth factors and cytokines, such as platelet-derived growth factor-BB (PDGF-BB). Here we used a genetic cell model to delineate defined signaling pathways that enhance collagen gel contraction downstream of ligand-stimulated platelet-derived growth factor receptor-β (PDGF-Rβ). Our data show that PDGF BB-enhanced activations of phosphatidylinositol 3′-kinase (PI3K) and phospholipase Cγ (PLCγ) were necessary for PDGF-enhanced collagen gel contraction. Importantly, other defined signaling pathways down-stream of PDGF-Rβ were, however, dispensable. The decisive roles for PI3K and PLCγ were corroborated by experiments using selective inhibitors. Furthermore, we show that de-phosphorylation and thereby activation of cofilin that is important for the turnover of actin filaments, is depended on PI3K and PLCγ down-stream of PDGF-Rβ. Moreover, inhibition of protein kinase C (PKC) by GÖ6976 and bisindolylmaleimide-II abolished cofilin de-phosphorylation, as well as PDGF-enhanced contraction. In contrast, activation of the PKC protein family by 4β-phorbol 12-myristate 13-acetate (PMA) did not accelerate collagen gel contraction although it induced long-term cofilin de-phosphorylation, showing the need of a dynamic control of cofilin de-phosphorylation for PDGF-enhanced collagen gel contraction. Taken together, our data point to the involvement of a PI3K/PLCγ-PKC-cofilin pathway in both PDGF-enhanced cofilin de-phosphorylation and PDGF-enhanced collagen gel contraction.

Tumor barrier function in carcinoma represents a major challenge to treatment and is therefore an attractive target for increasing drug delivery. Variables related to tumor barrier include aberrant blood vessels, high interstitial fluid pressure, and the composition and structure of the extracellular matrix. One of the proteins associated with dense extracellular matrices is fibromodulin, a collagen fibrillogenesis modulator expressed in tumor stroma but scarce in normal loose connective tissues. Here, we investigated the effects of fibromodulin on stroma ECM in a syngeneic murine colon carcinoma model. We show that fibromodulin deficiency decreased collagen fibril thickness but glycosaminoglycan content and composition were unchanged. Furthermore, vascular density, pericyte coverage and macrophage amount were unaffected. Fibromodulin can therefore be a unique effector of dense collagen matrix assembly in tumor stroma and, without affecting other major matrix components or the cellular composition, can function as a main agent in tumor barrier function.

Imatinib causes increased turnover of stromal collagen, reduces collagen fibril diameter, enhances extracellular fluid turnover and lowers interstitial fluid pressure (IFP) in the human colonic carcinoma KAT-4/HT-29 (KAT-4) xenograft model. We compared the effects of imatinib on oxygen levels, vascular morphology and IFP in three experimental tumor models differing in their content of a collagenous extracellular matrix. Neither the KAT4 and CT-26 colonic carcinoma models, nor B16BB melanoma expressed PDGF β-receptors in the malignant cells. KAT-4 tumors exhibited a well-developed ECM in contrast to the other two model systems. The collagen content was substantially higher in KAT-4 than in CT-26, while collagen was not detectable in B16BB tumors. The pO2 was on average 5.4, 13.9 and 19.3 mmHg in KAT-4, CT-26 and B16BB tumors, respectively. Treatment with imatinib resulted in similar pO2-levels in all three tumor models but only in KAT-4 tumors did the increase reach statistical significance. It is likely that after imatinib treatment the increase in pO2 in KAT-4 tumors is caused by increased blood flow due to reduced vascular resistance. This notion is supported by the significant reduction observed in IFP in KAT-4 tumors after imatinib treatment. Vessel area varied between 4.5 and 7% in the three tumor models and was not affected by imatinib treatment. Imatinib had no effect on the fraction of proliferating cells, whereas the fraction of apoptotic cells increased to a similar degree in all three tumor models. Our data suggest that the effects of imatinib on pO2-levels depend on a well-developed ECM and provide further support to the suggestion that imatinib acts by causing interstitial stroma cells to produce a less dense ECM, which would in turn allow for an increased blood flow. The potential of imatinib treatment to render solid tumors more accessible to conventional treatments would therefore depend on the degree of tumor desmoplasia.

A typical obstacle to cancer therapy is the limited distribution of low molecular weight anticancer drugs within the carcinoma tissue. In experimental carcinoma, imatinib (STI571) increases efficacy of synchronized chemotherapy, reduces tumor interstitial fluid pressure, and increases interstitial fluid volume. STI571 also increases the water-perfusable fraction in metastases from human colorectal adenocarcinomas. Because the mechanism(s) behind these effects have not been fully elucidated, we investigated the hypothesis that STI571 alters specific properties of the stromal extracellular matrix. We analyzed STI571-treated human colorectal KAT-4/HT-29 experimental carcinomas, known to have a well-developed stromal compartment, for solute exchange and glycosaminoglycan content, as well as collagen content, structure, and synthesis. MRI of STI571-treated KAT-4/HT-29 experimental carcinomas showed a significantly increased efficacy in dynamic exchanges of solutes between tumor interstitium and blood. This effect was paralleled by a distinct change of the stromal collagen network architecture, manifested by a decreased average collagen fibril diameter, and increased collagen turnover. The glycosaminoglycan content was unchanged. Furthermore, the apparent effects on the stromal cellular composition were limited to a reduction in an NG2-positive stromal cell population. The current data support the hypothesis that the collagen network architecture influences the dynamic exchanges of solutes between blood and carcinoma tissue. It is conceivable that STI571 reprograms distinct nonvascular stromal cells to produce a looser extracellular matrix, ultimately improving transport characteristics for traditional chemotherapeutic agents. Mol Cancer Ther; 15(10); 2455–64. ©2016 AACR.

Tumor stroma is similar to the connective tissue of chronic inflammation. The extracellular matrix of tumors is formed by cancer-associated fibroblasts that also modulate the inflammatory response. We studied the ability of oral keratinocytes (NOK) and oral squamous cell carcinoma cells (SCC) to induce an innate immune response in fibroblasts. Co-cultures with fibroblasts in collagen gels and keratinocytes in inserts were used. Pentraxin 3 (PTX3) was used as an indicator of an innate immune response. SCC and NOK up-regulated fibroblast mRNA expression and protein release of PTX3. mRNA levels were more pronounced in cultures with malignant cells. The induction of PTX3 was abrogated by an interleukin-1 receptor antagonist Keratinocytes have the capacity to induce an interleukin-1-dependent innate immune response by fibroblasts in vitro. This could be important for subsequent fibroblast modulation of the inflammatory reaction in non-malignant and malignant disease processes.