Inhibition of CXCR4 Enhances the Efficacy of Radiotherapy in Metastatic Prostate Cancer Models.

Radiotherapy (RT) is a standard treatment for patients with advanced prostate cancer (PCa). Previous preclinical studies showed that SDF1α/CXCR4 axis could mediate PCa metastasis (most often to the bones) and cancer resistance to RT. We found high levels of expression for both SDF1α and its receptor CXCR4 in primary and metastatic PCa tissue samples. In vitro analyses using PCa cells revealed an important role of CXCR4 in cell invasion but not radiotolerance. Pharmacologic inhibition of CXCR4 using AMD3100 showed no efficacy in orthotopic primary and bone metastatic PCa models. However, when combined with RT, AMD3100 potentiated the effect of local single-dose RT (12 Gy) in both models. Moreover, CXCR4 inhibition also reduced lymph node metastasis from primary PCa. Notably, CXCR4 inhibition promoted the normalization of bone metastatic PCa vasculature and reduced tissue hypoxia. In conclusion, the SDF1α/CXCR4 axis is a potential therapeutic target in metastatic PCa patients treated with RT.

The effects of valve leaflet mechanics on lymphatic pumping assessed using numerical simulations.

The lymphatic system contains intraluminal leaflet valves that function to bias lymph flow back towards the heart. These valves are present in the collecting lymphatic vessels, which generally have lymphatic muscle cells and can spontaneously pump fluid. Recent studies have shown that the valves are open at rest, can allow some backflow, and are a source of nitric oxide (NO). To investigate how these valves function as a mechanical valve and source of vasoactive species to optimize throughput, we developed a mathematical model that explicitly includes Ca2+ -modulated contractions, NO production and valve structures. The 2D lattice Boltzmann model includes an initial lymphatic vessel and a collecting lymphangion embedded in a porous tissue. The lymphangion segment has mechanically-active vessel walls and is flanked by deformable valves. Vessel wall motion is passively affected by fluid pressure, while active contractions are driven by intracellular Ca2+ fluxes. The model reproduces NO and Ca2+ dynamics, valve motion and fluid drainage from tissue. We find that valve structural properties have dramatic effects on performance, and that valves with a stiffer base and flexible tips produce more stable cycling. In agreement with experimental observations, the valves are a major source of NO. Once initiated, the contractions are spontaneous and self-sustained, and the system exhibits interesting non-linear dynamics. For example, increased fluid pressure in the tissue or decreased lymph pressure at the outlet of the system produces high shear stress and high levels of NO, which inhibits contractions. On the other hand, a high outlet pressure opposes the flow, increasing the luminal pressure and the radius of the vessel, which results in strong contractions in response to mechanical stretch of the wall. We also find that the location of contraction initiation is affected by the extent of backflow through the valves.

A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma.

Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region. We also describe how to apply state-of-the-art intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor-stroma interaction assays can be applied, and the model can be used to test the efficacy of novel therapeutic approaches. By studying the disease from every angle, this model offers the potential to unravel the basic biological underpinnings of NF2 and to develop novel therapeutics to control this devastating disease. Our protocol can be adapted to study other diseases within the CPA, including meningiomas, lipomas, vascular malformations, hemangiomas, epidermoid cysts, cerebellar astrocytomas, and metastatic lesions. The entire surgical procedure takes ~45 min per mouse and allows for subsequent longitudinal imaging, as well as neurological and hearing assessment, for up to 2 months.

Vascular beds maintain pancreatic tumour explants for ex vivo drug screening.

Our understanding of cancer progression or response to therapies would benefit from benchtop, tissue-level assays that preserve the biology and anatomy of human tumours ex vivo. We present a methodology for maintaining patient tumour samples ex vivo for the purpose of drug testing in a clinical setting. The harvested tumour biopsy, excised from mice or patients, is integrated into a support tissue that includes stroma and vasculature. This support tissue preserves tumour histoarchitecture and relevant expression profiles, and tumour tissues cultured using this system display different sensitivities to chemotherapeutics compared with tumour explants with no supporting tissue. The methodology is more rapid than patient-derived xenograft models, easy to implement, and amenable to high-throughput assays, making it an attractive tool for in vitro drug screening or for the guidance of patient-specific chemotherapies.

Secretory leukocyte protease inhibitor (SLPI) as a potential target for inhibiting metastasis of triple-negative breast cancers.

SLPI has been implicated in the progression and metastasis of certain cancers. However, the effects of SLPI seem to be tumor-specific and the mechanisms remain poorly defined. Here, we demonstrate that highly metastatic, triple-negative breast cancer (TNBC) 4T1 cells secreted more SLPI compared to their non-metastatic counterparts. Furthermore, SLPI secretion directly correlated with spontaneous lung metastasis from 4T1 tumors orthotopically implanted in mice. Consistent with our experimental results, we also found that higher SLPI expression levels correlate with worse clinical outcome in basal/TNBC patients. Using high-throughput screening we identified a novel compound, C74, which significantly inhibits SLPI secretion. C74 administration in our mouse model slows the growth of primary 4T1 tumors and inhibits their dissemination to the lung. We also discovered that SLPI physically interacts with the retinoblastoma tumor suppressor protein (Rb) and releases FoxM1 from the Rb-FoxM1 complex, which may activate FoxM1 target genes involved in breast cancer metastasis.

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases.

The survival benefit of anti-vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.

Self-assembly of vascularized tissue to support tumor explants in vitro.

Testing the efficacy of cancer drugs requires functional assays that recapitulate the cell populations, anatomy and biological responses of human tumors. Although current animal models and in vitro cell culture platforms are informative, they have significant shortcomings. Mouse models can reproduce tissue-level and systemic responses to tumor growth and treatments observed in humans, but xenografts from patients often do not grow, or require months to develop. On the other hand, current in vitro assays are useful for studying the molecular bases of tumorigenesis or drug activity, but often lack the appropriate in vivo cell heterogeneity and natural microenvironment. Therefore, there is a need for novel tools that allow rapid analysis of patient-derived tumors in a robust and representative microenvironment. We have developed methodology for maintaining harvested tumor tissue in vitro by placing them in a support bed with self-assembled stroma and vasculature. The harvested biopsy or tumor explant integrates with the stromal bed and vasculature, providing the correct extracellular matrix (collagen I, IV, fibronectin), associated stromal cells, and a lumenized vessel network. Our system provides a new tool that will allow ex vivo drug-screening and can be adapted for the guidance of patient-specific therapeutic strategies.

Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting.

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity, and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic.

Implantable tissue isolation chambers for analyzing tumor dynamics in vivo.

Recruitment of new blood vessels from the surrounding tissue is central to tumor progression and involves a fundamental transition of the normal, organized vasculature into a dense disarray of vessels that infiltrates the tumor. At present, studying the co-development of the tumor and recruited normal tissue is experimentally challenging because many of the important events occur rapidly and over short length scales in a dense three-dimensional space. To overcome these experimental limitations, we partially confined tumors within biocompatible and optically clear tissue isolation chambers (TICs) and implanted them in mice to create a system that is more amenable to microscopic analysis. Our goal was to integrate the tumor into a recruited host tissue - complete with vasculature - and demonstrate that the system recapitulates relevant features of the tumor microenvironment. We show that the TICs allow clear visualization of the cellular events associated with tumor growth and progression at the host-tumor interface including cell infiltration, matrix remodeling and angiogenesis. The tissue within the chamber is viable for more than a month, and the process is robust in both the skin and brain. Treatment with losartan, an angiotensin II receptor antagonist, decreased the collagen density and fiber length in the TIC, consistent with the known activity of this drug. We further show that collagen fibers display characteristic tumor signatures and play a central role in angiogenesis, guiding the migration of tethered endothelial sprouts. The methodology combines accessible methods of microfabrication with animal models and will enable more informative studies of the cellular mechanisms of tumor progression.

Extracellular matrix fibronectin initiates endothelium-dependent arteriolar dilatation via the heparin-binding, matricryptic RWRPK sequence of the first type III repeat of fibrillar fibronectin.

KEY POINTS: The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms. In addition to the many metabolic signals that mediate this vasodilatation, we show here that the extracellular matrix protein fibronectin also contributes to the response. This vasodilatory signal requires the heparin-binding matricryptic RWRPK sequence in the first type III repeat of fibrillar fibronectin. The fibronectin-dependent component of the integrated muscle contraction-dependent arteriolar vasodilatation is coupled through an endothelial cell-dependent signalling pathway. Recent studies in contracting skeletal muscle have shown that functional vasodilatation in resistance arterioles has an endothelial cell (EC)-dependent component, and, separately have shown that the extracellular matrix protein fibronectin (FN) contributes to functional dilatation in these arterioles. Here we test the hypotheses that (i) the matricryptic heparin-binding region of the first type III repeat of fibrillar FN (FNIII1H) mediates vasodilatation, and (ii) this response is EC dependent. Engineered FN fragments with differing (defined) heparin- and integrin-binding capacities were applied directly to resistance arterioles in cremaster muscles of anaesthetized (pentobarbital sodium, 65 mg kg(-1)) mice. Both FNIII1H,8-10 and FNIII1H induced dilatations (12.2 ± 1.7 µm, n = 12 and 17.2 ± 2.4 µm, n = 14, respectively) whereas mutation of the active sequence (R(613) WRPK) of the heparin binding region significantly diminished the dilatation (3.2 ± 1.8 µm, n = 10). Contraction of skeletal muscle fibres via electrical field stimulation produced a vasodilatation (19.4 ± 1.2 µm, n = 12) that was significantly decreased (to 7.0 ± 2.7 µm, n = 7, P < 0.05) in the presence of FNIII1Peptide 6, which blocks extracellular matrix (ECM) FN and FNIII1H signalling. Furthermore, FNIII1H,8-10 and FNIII1H applied to EC-denuded arterioles failed to produce any dilatation indicating that endothelium was required for the response. Finally, FNIII1H significantly increased EC Ca(2+) (relative fluorescence 0.98 ± 0.02 in controls versus 1.12 ± 0.05, n = 17, P < 0.05). Thus, we conclude that ECM FN-dependent vasodilatation is mediated by the heparin-binding (RWRPK) sequence of FNIII1 in an EC-dependent manner. Importantly, blocking this signalling sequence decreased the dilatation to skeletal muscle contraction, indicating that there is a physiological role for this FN-dependent mechanism.

Pre-exposure to adenosine, acting via A(2A) receptors on endothelial cells, alters the protein kinase A dependence of adenosine-induced dilation in skeletal muscle resistance arterioles.

Adenosine (ADO) is an endogenous vasodilatory purine widely recognized to be a significant contributor to functional hyperaemia. Despite this, many aspects of the mechanisms by which ADO induces dilation in small resistance arterioles are not established, or appear contradictory. These include: identification of the primary receptor subtype; its location on endothelial (EC) or vascular smooth muscle cells; whether ADO acts on KATP channels in these resistance vessels; and the contribution of cAMP/protein kinase A (PKA) signalling to the response. In intravital microscopy studies of intact or EC-denuded skeletal muscle arterioles, we show that ADO acts via A2A receptors located on ECs to produce vasodilation via activation of KATP channels located on vascular smooth muscle cells. Importantly, we found that the signalling pathway involves cAMP as expected, but that a requirement for PKA activation is demonstrable only if the vessel is not pre-exposed to ADO. That is, PKA-dependent signalling varies with pre-exposure to ADO. Further, we show that PKA activation alone is not sufficient to dilate these arterioles; an additional EC calcium-dependent signalling mechanism is required for vasodilation to ADO. The ability of arterioles in situ to respond to occupancy of a specific receptor by utilizing different cell signalling pathways under different conditions to produce the same response allows the arteriole to respond to key homeostatic requirements using more than a single signalling mechanism. Clearly, this is likely to be physiologically advantageous, but the role for this signalling flexibility in the integrated arteriolar response that underlies functional hyperaemia will require further exploration.