Angiocrine signals regulate quiescence and therapy resistance in bone metastasis.

Bone provides supportive microenvironments for hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and is a frequent site of metastasis. While incidences of bone metastases increase with age, the properties of the bone marrow microenvironment that regulate dormancy and reactivation of disseminated tumor cells (DTCs) remain poorly understood. Here, we elucidate the age-associated changes in the bone secretome that trigger proliferation of HSCs, MSCs, and DTCs in the aging bone marrow microenvironment. Remarkably, a bone-specific mechanism involving expansion of pericytes and induction of quiescence-promoting secretome rendered this proliferative microenvironment resistant to radiation and chemotherapy. This bone-specific expansion of pericytes was triggered by an increase in PDGF signaling via remodeling of specialized type H blood vessels in response to therapy. The decline in bone marrow pericytes upon aging provides an explanation for loss of quiescence and expansion of cancer cells in the aged bone marrow microenvironment. Manipulation of blood flow - specifically, reduced blood flow - inhibited pericyte expansion, regulated endothelial PDGF-B expression, and rendered bone metastatic cancer cells susceptible to radiation and chemotherapy. Thus, our study provides a framework to recognize bone marrow vascular niches in age-associated increases in metastasis and to target angiocrine signals in therapeutic strategies to manage bone metastasis.

Loss of Pericytes in Radiation Necrosis after Glioblastoma Treatments.

Radiation necrosis (RN) in brain tumor patients is often symptomatic, persistent without immediate resolution, and confused with tumor recurrence. Cerebral vascular pericytes are essential for endothelial function, vascular integrity, and angiogenesis. In this study, we showed that the loss of pericytes is involved in the pathogenesis of RN. From a brain tumor tissue repository, we identified three patients since 2011 with pathologically confirmed RN after the standard treatment for glioblastoma (GBM). The RN and their preradiation GBM tissues were serially processed for Western blotting using cell-type-specific antibodies against endothelial (CD31, active RhoA), pericyte [platelet-derived growth factor receptor-beta (PDGFR-ß)], alpha-smooth muscle actin (α-SMA), astrocyte (GFAP), myelin sheath protein (MBP), and microglial markers (Iba1). Normal brain tissues from a brain bank were used as normal controls. The expressions of PDGFR-ß and α-SMA were remarkably reduced in the RN, compared to those of GBM. However, the levels of CD31 or RhoA were not different between the two groups, which suggest that there was no change in the number of endothelial cells or their cytoskeletal assembly. The RN tissues showed a decreased ratio of pericyte/endothelial markers and an increased level of Iba1 compared to the GBM and even to the normal brain. The levels of GFAP and MBP were not changed in the RN. In the histopathology, the RN tissues showed a loss of markers (PDGFR-ß), whereas the GBM tissues had abundant expression of the markers. The loss of pericytes and vascular smooth muscle cells, and the unsupported endothelial cells might be the cause of the leaky blood-brain barrier and tissue necrosis.

In-vivo quantification of the revascularization of a human acellular dermis seeded with EPCs and MSCs in co-culture with fibroblasts and pericytes in the dorsal chamber model in pre-irradiated tissue.

INTRODUCTION: Transplantation of a cell-seeded graft may improve wound healing after radiotherapy. However, the survival of the seeded cells depends on a rapid vascularization of the graft. Co-culturing of adult stem cells may be a promising strategy to accelerate the vessel formation inside the graft. Thus, we compared the in vivo angiogenic potency of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) using dorsal skinfold chambers and intravital microscopy. MATERIALS AND METHODS: Cells were isolated from rat bone marrow and adipose tissue and characterized by immunostaining and flow cytometry. Forty-eight rats received a dorsal skinfold chamber and were divided into 2 main groups, irradiated and non-irradiated. Each of these 2 groups were further subdivided into 4 groups: unseeded matrices, matrices + fibroblasts + pericytes, matrices + fibroblasts + pericytes + MSCs and matrices + fibroblasts + pericytes + EPCs. Vessel densities were quantified semi-automatically using FIJI. RESULTS: Fibroblasts + pericytes - seeded matrices showed a significantly higher vascular density in all groups with an exception of non-irradiated rats at day 12 compared to unseeded matrices. Co-seeding of MSCs increased vessel densities in both, irradiated and non-irradiated groups. Co-seeding with EPCs did not result in an increase of vascularization in none of the groups. DISCUSSION: We demonstrated that the pre-radiation treatment led to a significant decreased vascularization of the implanted grafts. The augmentation of the matrices with fibroblasts and pericytes in co-culture increased the vascularization compared to the non-seeded matrices. A further significant enhancement of vessel ingrowth into the matrices could be achieved by the co-seeding with MSCs in both, irradiated and non-irradiated groups.

High-Dose, Single-Fraction Irradiation Rapidly Reduces Tumor Vasculature and Perfusion in a Xenograft Model of Neuroblastoma.

PURPOSE: To characterize the effects of high-dose radiation therapy (HDRT) on neuroblastoma tumor vasculature, including the endothelial cell (EC)-pericyte interaction as a potential target for combined treatment with antiangiogenic agents. METHODS AND MATERIALS: The vascular effects of radiation therapy were examined in a xenograft model of high-risk neuroblastoma. In vivo 3-dimensional contrast-enhanced ultrasonography (3D-CEUS) imaging and immunohistochemistry (IHC) were performed. RESULTS: HDRT significantly reduced tumor blood volume 6 hours after irradiation compared with the lower doses used in conventionally fractionated radiation. There was a 63% decrease in tumor blood volume after 12-Gy radiation compared with a 24% decrease after 2 Gy. Analysis of tumor vasculature by lectin angiography showed a significant loss of small vessel ends at 6 hours. IHC revealed a significant loss of ECs at 6 and 72 hours after HDRT, with an accompanying loss of immature and mature pericytes at 72 hours. CONCLUSIONS: HDRT affects tumor vasculature in a manner not observed at lower doses. The main observation was an early reduction in tumor perfusion resulting from a reduction of small vessel ends with a corresponding loss of endothelial cells and pericytes.

Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer.

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)-based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8(+) T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8(+) T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials.

Combination of vessel-targeting agents and fractionated radiation therapy: the role of the SDF-1/CXCR4 pathway.

PURPOSE: To investigate vascular responses during fractionated radiation therapy (F-RT) and the effects of targeting pericytes or bone marrow-derived cells (BMDCs) on the efficacy of F-RT. METHODS AND MATERIALS: Murine prostate TRAMP-C1 tumors were grown in control mice or mice transplanted with green fluorescent protein-tagged bone marrow (GFP-BM), and irradiated with 60 Gy in 15 fractions. Mice were also treated with gefitinib (an epidermal growth factor receptor inhibitor) or AMD3100 (a CXCR4 antagonist) to examine the effects of combination treatment. The responses of tumor vasculatures to these treatments and changes of tumor microenvironment were assessed. RESULTS: After F-RT, the tumor microvascular density (MVD) was reduced; however, the surviving vessels were dilated, incorporated with GFP-positive cells, tightly adhered to pericytes, and well perfused with Hoechst 33342, suggesting a more mature structure formed primarily via vasculogenesis. Although the gefitinib+F-RT combination affected the vascular structure by dissociating pericytes from the vascular wall, it did not further delay tumor growth. These tumors had higher MVD and better vascular perfusion function, leading to less hypoxia and tumor necrosis. By contrast, the AMD3100+F-RT combination significantly enhanced tumor growth delay more than F-RT alone, and these tumors had lower MVD and poorer vascular perfusion function, resulting in increased hypoxia. These tumor vessels were rarely covered by pericytes and free of GFP-positive cells. CONCLUSIONS: Vasculogenesis is a major mechanism for tumor vessel survival during F-RT. Complex interactions occur between vessel-targeting agents and F-RT, and a synergistic effect may not always exist. To enhance F-RT, using CXCR4 inhibitor to block BM cell influx and the vasculogenesis process is a better strategy than targeting pericytes by epidermal growth factor receptor inhibitor.

Alterations in daily sequencing of axitinib and fractionated radiotherapy do not affect tumor growth inhibition or pathophysiological response.

A variety of antiangiogenic strategies have proven effective in preclinical tumor models, either as single agents or in combination with radiation. Clinical gains have been relatively modest, however, and questions remain regarding optimal scheduling. The objectives of the current work were to evaluate whether the sequencing of acute treatment critically affects tumor pathophysiological and therapeutic response. Axitinib (Pfizer Global Research & Development), an inhibitor that predominantly targets vascular endothelial growth factor receptors, was administered either before or after each daily radiation fraction in two human prostate xenograft tumor models. Tumors were frozen at sequential times to monitor changes in (1) vascular spacing, (2) pericyte and basement membrane coverage, and (3) hypoxia. Although similar reductions in blood vessel counts were observed with each tumor model, tumor vasculature was not functionally normalized. Instead, tumor hypoxia increased, accompanied by a progressive dissociation of pericytes and basement membranes. Ultimately, tumor growth inhibition was found to be equivalent for each of the combination schedules. These studies illustrate a clear advantage to combining axitinib with fractionated therapy but argue against an acute radiosensitization or radioprotection of either the tumor cells or tumor vasculature. Instead, post- and preirradiation daily drug administration serve equally well in supplementing the response to radiotherapy.

Antiangiogenic therapy: creating a unique "window" of opportunity.

Antiangiogenic therapy for solid tumors clearly destroys tumor vasculature and reduces tumor growth. As an unexpected bonus, drugs that neutralize VEGF signaling generate a "normalization window" for tumor vasculature. This occurs via the recruitment of pericytes to the tumor vasculature, an effect associated with the transient stabilization of vessels and improved oxygen delivery to hypoxic zones. The normalization process is mediated by angiopoietin-1 and matrix metalloproteinases and creates a window of opportunity for improved sensitivity to ionizing radiation and the delivery of chemotherapeutic drugs.

The effects of high ambient glucose on the radiosensitivity of retinal microvascular endothelial cells and pericytes.

PURPOSE: The metabolic peturbations of diabetes cause functional and structural changes in the retinal microvasculature which are termed diabetic retinopathy. Exposure of the eye to ionising radiation results in retinal vascular damage with a clinical manifestation known as radiation retinopathy. Anecdotal studies have suggested that exposure to even low levels of ionising radiation may accelerate development of pathological changes in the retinal vessels of patients with diabetes. This in vitro study was designed to test the hypothesis that the combination of a high ambient glucose environment (mimicking hyperglycaemia and diabetes) along with exposure to ionising radiation would result in more accentuated damage to cultured retinal vascular cells. METHODS: Retinal microvascular endothelial cells and pericytes were propagated for 5 days in either 5 mM (euglycaemia) or 15 mM (hyperglycaemia) glucose. Cells were irradiated with 250, 500 or 1000 cGy of ionising radiation using a 6 MV beam photon accelerator which was used for radiotherapy. Similarly treated but unirradiated cells were used as controls. DNA damage was assessed using the single-cell gel electrophoresis (comet) assay. RESULTS: Unirradiated control cells pre-exposed to glucose at either 5 mM or 15 mM for 5 days showed no significant difference in mean percentage tail DNA representing damage. However, in both pericytes and endothelial cells exposed to ionising radiation, cells cultured in 15 mM glucose showed significantly higher levels of DNA damage compared with those cultured in 5 mM glucose, with maximal differences being seen at the higher radiation doses (500 and 1000 cGy). CONCLUSIONS: This study has demonstrated that retinal microvascular cells cultured in high glucose express more DNA damage when exposed ionising radiation. These findings have important implications for the management of patients with diabetes if they require radiotherapy for neoplastic disease.