James Monslow

Tumor-promoting desmoplasia is disrupted by depleting FAP-expressing stromal cells

Malignant cells drive the generation of a desmoplastic and immunosuppressive tumor microenvironment. Cancer-associated stromal cells (CASC) are a heterogeneous population that provides both negative and positive signals for tumor cell growth and metastasis. Fibroblast activation protein (FAP) is a marker of a major subset of CASCs in virtually all carcinomas. Clinically, FAP expression serves as an independent negative prognostic factor for multiple types of human malignancies. Prior studies established that depletion of FAP(+) cells inhibits tumor growth by augmenting antitumor immunity. However, the potential for immune-independent effects on tumor growth have not been defined. Herein, we demonstrate that FAP(+) CASCs are required for maintenance of the provisional tumor stroma because depletion of these cells, by adoptive transfer of FAP-targeted chimeric antigen receptor (CAR) T cells, reduced extracellular matrix proteins and glycosaminoglycans. Adoptive transfer of FAP-CAR T cells also decreased tumor vascular density and restrained growth of desmoplastic human lung cancer xenografts and syngeneic murine pancreatic cancers in an immune-independent fashion. Adoptive transfer of FAP-CAR T cells also restrained autochthonous pancreatic cancer growth. These data distinguish the function of FAP(+) CASCs from other CASC subsets and provide support for further development of FAP(+) stromal cell-targeted therapies for the treatment of solid tumors.

Cardiovascular protection by ApoE and ApoE-HDL linked to suppression of ECM gene expression and arterial stiffening.

Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing high-density lipoprotein (apoE-HDL) maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE null mice. This stiffening can be reduced by administration of the lysyl oxidase inhibitor BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics and that this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol.

Hyaluronan - a functional and structural sweet spot in the tissue microenvironment.

Transition from homeostatic to reactive matrix remodeling is a fundamental adaptive tissue response to injury, inflammatory disease, fibrosis, and cancer. Alterations in architecture, physical properties, and matrix composition result in changes in biomechanical and biochemical cellular signaling. The dynamics of pericellular and extracellular matrices, including matrix protein, proteoglycan, and glycosaminoglycan modification are continually emerging as essential regulatory mechanisms underlying cellular and tissue function. Nevertheless, the impact of matrix organization on inflammation and immunity in particular and the consequent effects on tissue healing and disease outcome are arguably under-studied aspects of adaptive stress responses. Herein, we review how the predominant glycosaminoglycan hyaluronan (HA) contributes to the structure and function of the tissue microenvironment. Specifically, we examine the evidence of HA degradation and the generation of biologically active smaller HA fragments in pathological settings in vivo. We discuss how HA fragments versus nascent HA via alternate receptor-mediated signaling influence inflammatory cell recruitment and differentiation, resident cell activation, as well as tumor growth, survival, and metastasis. Finally, we discuss how HA fragmentation impacts restoration of normal tissue function and pathological outcomes in disease.

Cyclooxygenase-2 in Endothelial and Vascular Smooth Muscle Cells Restrains Atherogenesis in Hyperlipidemic Mice

Background— Placebo-controlled trials of nonsteroidal anti-inflammatory drugs selective for inhibition of cyclooxygenase-2 (COX-2) reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages. Methods and Results— In the present study, selective depletion of COX-2 in vascular smooth muscle cells and endothelial cells depressed biosynthesis of prostaglandin I2 and prostaglandin E2, elevated blood pressure, and accelerated atherogenesis in Ldlr knockout mice. Deletion of COX-2 in vascular smooth muscle cells and endothelial cells coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, &agr;-smooth muscle actin, and matrix-rich fibrosis was also apparent in lesions of the mutants. Conclusions— Although atherogenesis is accelerated in global COX-2 knockouts, consistent with evidence of risk transformation during chronic nonsteroidal anti-inflammatory drug administration, this masks the contrasting effects of enzyme depletion in macrophages versus vascular smooth muscle cells and endothelial cells. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk.

Myeloid cell microsomal prostaglandin E synthase-1 fosters atherogenesis in mice

Significance Inhibitors of microsomal prostaglandin E synthase-1 (mPGES-1) are being developed as analgesics. Although global depletion of mPGES-1 suggests they will be less likely to confer a cardiovascular hazard than NSAIDs selective for inhibition of COX-2, mPGES-1–derived PGE2 may have contrasting effects on discrete cellular components of the vasculature. mPGES-1 inhibition may result in substrate rediversion to other PG synthases, the products of which differ between cell types and exert contrasting biologies. Here, myeloid cell mPGES-1, reflective of the macrophage enzyme, promotes atherogenesis, fostering inflammation and oxidative stress. By contrast, mPGES-1 in endothelial and vascular smooth muscle cells has no discernable effect. Selective targeting of macrophage mPGES-1 may conserve cardiovascular benefit while avoiding adverse effects related to enzyme blockade in other tissues. Microsomal prostaglandin E synthase-1 (mPGES-1) in myeloid and vascular cells differentially regulates the response to vascular injury, reflecting distinct effects of mPGES-1–derived PGE2 in these cell types on discrete cellular components of the vasculature. The cell selective roles of mPGES-1 in atherogenesis are unknown. Mice lacking mPGES-1 conditionally in myeloid cells (Mac-mPGES-1-KOs), vascular smooth muscle cells (VSMC-mPGES-1-KOs), or endothelial cells (EC-mPGES-1-KOs) were crossed into hyperlipidemic low-density lipoprotein receptor-deficient animals. En face aortic lesion analysis revealed markedly reduced atherogenesis in Mac-mPGES-1-KOs, which was concomitant with a reduction in oxidative stress, reflective of reduced macrophage infiltration, less lesional expression of inducible nitric oxide synthase (iNOS), and lower aortic expression of NADPH oxidases and proinflammatory cytokines. Reduced oxidative stress was reflected systemically by a decline in urinary 8,12-iso-iPF2α-VI. In contrast to exaggeration of the response to vascular injury, deletion of mPGES-1 in VSMCs, ECs, or both had no detectable phenotypic impact on atherogenesis. Macrophage foam cell formation and cholesterol efflux, together with plasma cholesterol and triglycerides, were unchanged as a function of genotype. In conclusion, myeloid cell mPGES-1 promotes atherogenesis in hyperlipidemic mice, coincident with iNOS-mediated oxidative stress. By contrast, mPGES-1 in vascular cells does not detectably influence atherogenesis in mice. This strengthens the therapeutic rationale for targeting macrophage mPGES-1 in inflammatory cardiovascular diseases.

Matrix metalloproteinase-12 is an essential mediator of acute and chronic arterial stiffening.

Arterial stiffening is a hallmark of aging and risk factor for cardiovascular disease, yet its regulation is poorly understood. Here we use mouse modeling to show that matrix metalloproteinase-12 (MMP12), a potent elastase, is essential for acute and chronic arterial stiffening. MMP12 was induced in arterial smooth muscle cells (SMCs) after acute vascular injury. As determined by genome-wide analysis, the magnitude of its gene induction exceeded that of all other MMPs as well as those of the fibrillar collagens and lysyl oxidases, other common regulators of tissue stiffness. A preferential induction of SMC MMP12, without comparable effect on collagen abundance or structure, was also seen during chronic arterial stiffening with age. In both settings, deletion of MMP12 reduced elastin degradation and blocked arterial stiffening as assessed by atomic force microscopy and immunostaining for stiffness-regulated molecular markers. Isolated MMP12-null SMCs sense extracellular stiffness normally, indicating that MMP12 causes arterial stiffening by remodeling the SMC microenvironment rather than affecting the mechanoresponsiveness of the cells themselves. In human aortic samples, MMP12 levels strongly correlate with markers of SMC stiffness. We conclude that MMP12 causes arterial stiffening in mice and suggest that it functions similarly in humans.

Extracellular matrix directs phenotypic heterogeneity of activated fibroblasts

Activated fibroblasts are key players in the injury response, tumorigenesis, fibrosis, and inflammation. Dichotomous outcomes in response to varied stroma-targeted therapies in cancer emphasize the need to disentangle the roles of heterogeneous fibroblast subsets in physiological and pathophysiological settings. In wound healing, fibrosis, and myriad tumor types, fibroblast activation protein (FAP) and alpha-smooth muscle actin (αSMA) identify distinct, yet overlapping, activated fibroblast subsets. Prior studies established that FAPHi reactive fibroblasts and αSMAHi myofibroblasts can exert opposing influences in tumorigenesis. However, the factors that drive this phenotypic heterogeneity and the unique functional roles of these subsets have not been defined. We demonstrate that a convergence of ECM composition, elasticity, and transforming growth factor beta (TGF-β) signaling governs activated fibroblast phenotypic heterogeneity. Furthermore, FAPHi reactive fibroblasts and αSMAHi myofibroblasts exhibited distinct gene expression signatures and functionality in vitro, illuminating potentially unique roles of activated fibroblast subsets in tissue remodeling. These insights into activated fibroblast heterogeneity will inform the rational design of stroma-targeted therapies for cancer and fibrosis.

Cardiovascular Consequences of Prostanoid I Receptor Deletion in Microsomal Prostaglandin E Synthase-1-Deficient Hyperlipidemic Mice.

Background: Inhibitors of cyclooxygenase-2 alleviate pain and reduce fever and inflammation by suppressing the biosynthesis of prostacyclin (PGI2) and prostaglandin E2. However, suppression of these prostaglandins, particularly PGI2, by cyclooxygenase-2 inhibition or deletion of its I prostanoid receptor also predisposes to accelerated atherogenesis and thrombosis in mice. By contrast, deletion of microsomal prostaglandin E synthase 1 (mPGES-1) confers analgesia, attenuates atherogenesis, and fails to accelerate thrombogenesis, while suppressing prostaglandin E2, but increasing biosynthesis of PGI2. Methods: To address the cardioprotective contribution of PGI2, we generated mice lacking the I prostanoid receptor together with mPges-1 on a hyperlipidemic background (low-density lipoprotein receptor knockouts). Results: mPges-1 depletion modestly increased thrombogenesis, but this response was markedly further augmented by coincident deletion of the I prostanoid receptor (n=10–18). By contrast, deletion of the I prostanoid receptor had no effect on the attenuation of atherogenesis by mPGES-1 deletion in the low-density lipoprotein receptor knockout mice (n=17–21). Conclusions: Although suppression of prostaglandin E2 accounts for the protective effect of mPGES-1 deletion in atherosclerosis, augmentation of PGI2 is the dominant contributor to its favorable thrombogenic profile. The divergent effects on these prostaglandins suggest that inhibitors of mPGES-1 may be less likely to cause cardiovascular adverse effects than nonsteroidal anti-inflammatory drugs specific for inhibition of cyclooxygenase-2.

Identification of prognostic collagen signatures and potential therapeutic stromal targets in canine mammary gland carcinoma

Increasing evidence indicates that the tumor microenvironment plays a critical role in regulating the biologic behavior of breast cancer. In veterinary oncology, there is a need for improved prognostic markers to accurately identify dogs at risk for local and distant (metastatic) recurrence of mammary gland carcinoma and therefore would benefit from adjuvant therapy. Collagen density and fiber organization have been shown to regulate tumor progression in both mouse and human mammary tumors, with certain collagen signatures predicting poor outcomes in women with breast cancer. We hypothesized that collagen signatures in canine mammary tumor biopsies can serve as prognostic biomarkers and potential targets for treatment. We used second harmonic generation imaging to evaluate fibrillar collagen density, the presence of a tumor-stromal boundary, tumor associated collagen signatures (TACS) and individual collagen fiber characteristics (width, length and straightness) in grade I/II and grade III canine mammary tumors. Collagen density, as well as fiber width, length and straightness, were inversely correlated with patient overall survival time. Notably, grade III cases were less likely to have a tumor-stromal boundary and the lack of a boundary predicted poor outcome. Importantly, a lack of a defined tumor-stromal boundary and an increased collagen fiber width were associated with decreased survival even when tumor grade, patient stage, ovariohysterectomy status at the time of mammary tumor excision, and histologic evidence of lymphovascular invasion were considered in a multivariable model, indicating that these parameters could augment current methods to identify patients at high risk for local or metastatic progression/recurrence. Furthermore, these data, which identify for the first time, prognostic collagen biomarkers in naturally occurring mammary gland neoplasia in the dog, support the use of the dog as a translational model for tumor-stromal interactions in breast cancer.

Fibroblast activation protein augments progression and metastasis of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinomas (PDAs) are desmoplastic and can undergo epithelial-to-mesenchymal transition to confer metastasis and chemoresistance. Studies have demonstrated that phenotypically and functionally distinct stromal cell populations exist in PDAs. Fibroblast activation protein-expressing (FAP-expressing) cells act to enhance PDA progression, while α-smooth muscle actin myofibroblasts can restrain PDA. Thus, identification of precise molecular targets that mediate the protumorigenic activity of FAP+ cells will guide development of therapy for PDA. Herein, we demonstrate that FAP overexpression in the tumor microenvironment correlates with poor overall and disease-free survival of PDA patients. Genetic deletion of FAP delayed onset of primary tumor and prolonged survival of mice in the KPC mouse model of PDA. While genetic deletion of FAP did not affect primary tumor weight in advanced disease, FAP deficiency increased tumor necrosis and impeded metastasis to multiple organs. Lineage-tracing studies unexpectedly showed that FAP is not only expressed by stromal cells, but can also be detected in a subset of CD90+ mesenchymal PDA cells, representing up to 20% of total intratumoral FAP+ cells. These data suggest that FAP may regulate PDA progression and metastasis in cell-autonomous and/or non-cell-autonomous fashions. Together, these data support pursuing FAP as a therapeutic target in PDA.