extfieldsofstudy: Medicine Biology

text:
Desert Hedgehog/Patch2 Axis Contributes to Vascular Permeability and Angiogenesis in Glioblastoma

Glioblastoma multiforme (GBM) constitutes the most common and the most aggressive type of human tumors affecting the central nervous system. Prognosis remains dark due to the inefficiency of current treatments and the rapid relapse. Paralleling other human tumors, GBM contains a fraction of tumor initiating cells with the capacity to self-renew, initiate and maintain the tumor mass. These cells were found in close proximity to brain vasculature, suggesting functional interactions between brain tumor-initiating cells (BTICs) and endothelial cells within the so-called vascular niche. However, the mechanisms by which these cells impact on the endothelium plasticity and function remain unclear. Using culture of BTICs isolated from a cohort of 14 GBM patients, we show that BTICs secretome promotes brain endothelial cell remodeling in a VEGF-independent manner. Gene array analysis unmasked that BTICs-released factors drove the expression of Ptch2 in endothelial cells. Interestingly, BTICs produce desert hedgehog (DHH) ligand, enabling a paracrine DHH/Ptch2 signaling cascade that conveys elevated permeability and angiogenesis. Finally, DHH silencing in BTICs dramatically reduced tumor growth, as well as vascularization and intra-tumor permeability. Collectively, our data unveil a role for DHH in exacerbated tumor angiogenesis and permeability, which may ultimately favor glioblastoma growth, and thus place the DHH/Ptch2 nexus as a molecular target for novel therapies.

INTRODUCTION
Glioblastoma multiforme (GBM) is one of the most malignant cancers in adults with less than 5% of patients surviving more than 2 years and a median survival range between 15 and 18 months (Stupp et al., 2009). While current therapies include surgical resection, followed by a combined radio-chemotherapy regime (Stupp et al., 2009), relapse is almost inevitable and remains fatal. GBM are highly vascularized tumors, with large zone of hypoxia and necrosis and are highly addicted to pro-angiogenic signaling pathways, among which is the vascular endothelial growth factor A (VEGF-A; Olsson et al., 2006;Jain et al., 2007). Vascularization index, blood flux, and vascular permeability and edema are thus important parameters directly linked to tumor aggressiveness (Jain et al., 2007;Sorensen et al., 2009). Echoing this, the humanized anti-VEGF antibodies bevacizumab, as well as novel anti-angiogenic therapies were developed to prune and/or normalize the tumor blood supply (Carmeliet and Jain, 2011). However, recent randomized large clinical studies failed to demonstrate the effectiveness of such therapies in first line treatment in GBM (Chinot et al., 2014). In this context, identification of new molecular targets controlling endothelial plasticity is at the utmost importance, as it may open new routes for more efficient therapeutic strategies in GBM treatment.
The identification of cancer cells with stem-like properties has contributed to a better knowledge of molecular and cellular processes involved in tumor formation and treatment resistance (Singh et al., 2003(Singh et al., , 2004Bao et al., 2006). Indeed, GBM are believed to originate from such brain tumor-initiating cells (BTICs) with the unique ability to self-sustain and renew the tumor (Singh et al., 2003(Singh et al., , 2004Bao et al., 2006). Such studies have highlighted the importance of both cellular hierarchy and intratumoral heterogeneity in GBM (Verhaak et al., 2010;Brennan et al., 2013;Patel et al., 2014). Similarly to neural stem cells, BTICs are nested in a vascular niche, which provides a specific and confined microenvironment that favors bidirectional transference between endothelial cells from the vascular wall and cancer initiating cells from the tumor mass. In keeping with this, BTICs were characterized in the vicinity of brain endothelial cells, allowing close interaction and exchange of survival and fate signals (Calabrese et al., 2007;Galan-Moya et al., 2011). Several studies had also documented the ability of BTICs to transdifferentiate and integrate the tumor vasculature (Ricci-Vitiani et al., 2010;Wang et al., 2010;Cheng et al., 2013). However, the precise mechanism by which BTICs impact on endothelial plasticity remains poorly understood.
Among the putative candidates, the hedgehog (HH) pathway is known to regulate stem cell maintenance, differentiation and proliferation in embryonic development. Notably, the HH network coordinates the blood-brain barrier integrity in both development and adulthood in mouse models (Alvarez et al., 2011). In pathological conditions, HH aberrant activation was linked to tumor invasion, migration, and progression (Smyth et al., 1999;Evangelista et al., 2006;D' Amico et al., 2015;Qualtrough et al., 2015;Xu et al., 2015). Moreover, HH signaling was found implicated in acquired chemoresistance and cancer stem cell fate and properties (Bar et al., 2007;Justilien et al., 2014;Kong et al., 2014;Takebe et al., 2015).
In the present work, we investigated the effects of patientderived BTICs-secreted factors on long-term endothelial cell behavior. We found that BTICs secretome conveys endothelial remodeling in a VEGF/Src-independent manner. We discovered that BTICs-secreted factors induce the expression of the HH receptor Ptch2, in endothelial cells. Interestingly, BTICs produce desert hedgehog (DHH) ligand, enabling a paracrine DHH/Ptch2 signaling cascade that culminates in vascular leakage and increased angiogenic potential. Finally, DHH knockdown in BTICs dramatically reduced tumor formation and growth, as well as intra-tumor permeability. Thus, our data place DHH/Ptch2 nexus as a potential candidate for therapeutic intervention in GBM.

Animals
This study was carried out in accordance with the recommendations of local ethics committee (Paris Descartes university, Paris, France) and approved by French Ministry of Research (agreement number #00754.02). All experiments were performed in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123). Six weeks-old female BALB/c nude mice were obtained from Janvier Labs (Saint-Berthevin, France).
Stable knockdown of DHH was performed in retrovirallyinfected BTICs as described previously .
Briefly, HEK-293T cells were transfected with a mixture of pVSVg, pSPAX2, and pGIPZ-GFP or pGIPZ-GFP-DHH shRNA (Thermo Fisher Scientific, Illkirch, France). Supernatants were collected 2 days post-transfection, clarified by centrifugation and used to transduce BTICs. Transduction and knockdown efficiency were checked by flow cytometry and RT-PCR, respectively, prior implantation in animals.

Flow Cytometry Analysis
Ptch1 and Ptch2 expression were analyzed using an indirect immunofluorescence protocol. Cells were incubated with primary antibodies (Ptch2 SAB2101905 and Ptch1 AV44249, Sigma), washed three times with cold phosphate-buffered saline and further stained with the corresponding Alexa488-conjugated IgG (Life Technologies). Data were acquired on a FACScalibur (CellQuest software; BD) and analyzed using FlowJo (Ashland, OR, USA).

ELISA
Desert hedgehog (DHH) and Sonic hedgehog (SHH) secreted concentrations were analyzed by ELISA assay as described in (Azzi et al., 2010). Briefly, 50 µl of BTICs-CM were coated in a 96 well ELISA plate at 4°C overnight, and saturated (BSA 3%, 30 min). Primary antibodies (DHH SAB1407419 and SHH SAB2108581, Sigma), were then added, washed five times with a PBS-Tween 0.5% solution, and further stained with the corresponding HRPconjugated IgG. Plates were washed five times, and 50 µl of TMB substrate (Sigma) was added for 20 min in the dark. Absorbance was read at 650 nm. Recombinant DHH (Emelca Bioscience) and SHH (Sigma) were used to establish the standard curves.

Tubulogenesis Assay
Tubulogenesis assay was performed as previously described . Briefly matrigel was added to a 96-well plate and allowed to polymerize for 30 min at 37°C. hCMEC/D3 endothelial cells (10,000 cells/well) were pre-treated with BTICs-CM for 24 h, prior being seeded on top of matrigel. Images of at least five fields of view (FOV) per condition, randomly chosen, were acquired each hour over an 8-h period (Motic, AE21 microscope, Wetzlar, Germany). For tube formation and branches quantifications, images were processed using ImageJ software (NIH, Bethesda, MD, USA).

Sprouting Assay
Sprouting of hCMEC/D3 cells was assessed as described in (Wimmer et al., 2012). Briefly, 4000 Cytodex3 microcarrier beads (Sigma) were coated with collagen (BD Biosciences), and mixed with 10 6 hCMEC/D3 cells in warm EBM-2 medium. The mixture was incubated for 4 h at 37°C, with regular shaking (each 15 min). Coated beads were transferred to a new culture dish overnight to remove unattached cells, then washed with PBS and resuspended in a 2.5 mg/ml fibrinogen-aprotinin (0.15 Units/ml, Sigma) solution + bFGF2 (200 ng/ml, Sigma). The mixture was then distributed in eight wells Ibidi plate containing 0.625 U/mL of thrombin, and allowed to clot for 15 min at 37°C. EBM-2 medium or BTICs-CM were added on top of the fibrin matrix and sprouting allowed for 3 days.

Permeability Assays
In vitro and in vivo permeability assays were conducted as described in (Gavard and Gutkind, 2006;Le Guelte et al., 2012;Treps et al., 2015). Briefly, for in vitro assay, 1.10 5 hCMEC/D3 cells were seeded on 3 µm pore-size collagen-coated PTFE membranes (Costar, VWR, Fontenay-sous-Bois, France) for 3 days. Cells were then treated with BTICs-CM for 24 h, and permeability evaluated by FITC-dextran 40 kDa passage. Fluorescence was measured using the Fusion plate reader (Packard, San Diego, CA, USA). Data were normalized to untreated samples and expressed as the mean on three independent experiments. For in vivo miles assay, sterile Evans blue (1% in PBS, Sigma) was administrated by retro-orbital injection in ketamine/xylazine mixture (50 and 5 mg/kg) anesthetized animals. Mice were sacrificed 30 min later, and intra-tumor blue extravasation evaluated by absorbance. Results were normalized to skin punctures.

Xenografts and Tissue Staining
Brain tumor-initiating cells transduced with shRNA against DHH or non-silencing shRNA control were re-suspended in a mixture of PBS/matrigel (1:1), and injected subcutaneously (1.10 6 cells/injection) in the flank of BALB/c nude mice. Tumor formation and volume were assessed over the duration of the experiment and quantified using following the equation: volume = (width 2 × length)/2 formula, in a double-blind study. To evaluate intra-tumor hypoxic zones, pimonidazole was administrated at 30 mg/kg by intravenous injection, 1 h prior mice sacrifice and tumor extraction. Tumors were cryopreserved in OCT to be later processed for imaging analysis.
Tissue sections were obtained using Leica cryostat (Histology core-facility, Cochin Institute, Paris, France). Sections were fixed (PFA 4% for 30 min), permeabilized (Triton 0.5%, 10 min), saturated (BSA 3%, 2 h) and incubated with CD31 antibody (1/200, overnight at 4°C, BD Biosciences). Tissue samples were washed and further incubated with corresponding FITC-coupled secondary antibody for 1 h. Alternatively, sections were incubated with Hp-Dylight™549 conjugated antibody overnight at 4°C. Images of five different FOV of three different sections were acquired using Leica fluorescence microscope, and staining quantified using the ImageJ software.
All statistical analyses were performed on two or three independent experiments, using Prism software (GraphPad, La Jolla, CA, USA).

Secretome of Patient-Derived Glioblastoma-Initiating Cells Impacts on Brain Endothelial Cell Plasticity
While previous studies had highlighted the importance of the vascular niche in maintaining the BTICs population (Calabrese et al., 2007;Galan-Moya et al., 2011), how in turn BTICs might impact on endothelial fate is poorly documented. We thus explore the effects of patient-derived BTICs-secreted factors on endothelial cell remodeling. To this aim, confluent monolayers of human endothelial brain endothelial cells (hCMEC/D3) cells were exposed to BTICs-CM for 24 h. BTICs-CM induced tubule formation and branching to the same extent as VEGF-A. Interestingly, this effect was significantly higher than serumfree EBM-2 medium (Ctl) and was recapitulated in all 14 patient-derived BTICs tested ( Figure 1A). Accordingly, BTICs-CM promoted hCMEC/D3 sprouting from collagen-coated microcarriers ( Figure 1B). Indeed, both sprout length and sprout number were elevated, when compared to control conditions, albeit slightly lower than VEGF-A-treated conditions ( Figure 1B). As VEGF-A is a key driver of endothelial plasticity (Folkman, 2006;Gavard and Gutkind, 2006), we next assessed whether it was required for BTICs-CM-triggered angiogenic phenotype. While inhibiting VEGF-R2 tyrosine kinase activity resulted in a dramatic reduction in both sprouting length and number of sprouting cells upon VEGF-A stimulation, no overt changes were observed when exposed to BTICs-CM ( Figure 1C). Furthermore, BTICs-CM heightened endothelial permeability, as indicated by a two-fold increase of FITC-dextran passage (Figure 1D), an effect seen in all the 14 GBM patient-derived BTICs tested. Again, it is unlikely this effect is dependent on VEGF-A, as blocking Src kinase activity; the main downstream regulator of VEGF-A-induced permeability (Eliceiri et al., 1999;Gavard and Gutkind, 2006); only partially reduced the BTICs-CM-triggered endothelial permeability increase ( Figure 1E). Thus, our data suggest that BTICs-released factors induce endothelial cell remodeling in a VEGF-independent manner.

Ptch2 Receptor is Upregulated in BTIC-CM-Exposed Endothelial Cells
In order to gain further insights into the molecular mechanisms involved in BTICs-based action, a transcriptome analysis of endothelial cells exposed to four different BTICs-CM (#1, #2, #3, #4) was performed, and a heat map of up-and downregulated genes was generated (Figure 2A). Statistical analysis of shared genes with modified expression of at least two fold, and a p value of <0.05 was performed revealing seven genes of interest. PTCH2, DHRS3, ODZ2, EBF4, LPPR5, RHOU, and NXPH4 were found in silico to be significantly up-regulated in treated cells, when compared to untreated endothelial cells. An independent RNA analysis confirmed the up-regulation of: PTCH2, ODZ2, and LPPR5 ( Figure 2B). Among these three candidates, we next focused on PTCH2, one of the two HH receptors. Indeed, HH signaling is aberrantly activated in many cancers and contributes to both tumor (Smyth et al., 1999) and blood-brain barrier development (Alvarez et al., 2011). Moreover, HH signaling was reported to supervise tumorinitiating cell fate and phenotype (Bar et al., 2007;Justilien et al., 2014;Takebe et al., 2015). In this context, PTCH2 expression was observed in three different endothelial cell lines, only upon BTICs-CM challenge (Figure 2C), suggesting that this effect was not restricted to brain microcirculation. Further flow cytometry analysis showed that at various levels, Ptch2 was upregulated in response to a large panel of BTICs-CM (Figures 2D,E), suggesting that Ptch2 up-regulation is not a BTICs-type dependent phenomenon. Interestingly, PTCH1 expression remained unchanged in control and treated cells (Figures 2C,D). To address the functional role of Ptch2 in endothelial cells, RNA interference was employed to prevent BTICs-CM-driven up-regulation (Figures 2F-G). When Ptch2 was no longer induced, endothelial permeability and sprouting were significantly impaired (Figures 2H,I). Interestingly, Ptch1 siRNA failed to phenocopy Ptch2 silencing (Figures 2H,I). Finally, endothelial responses to VEGF-A were left intact in terms of sprouting and permeability (Figures 2H,I), reinforcing the VEGF-independent property of BTICs-secreted factors.

Desert Hedgehog Mediates Permeability and Angiogenesis in Brain Endothelial Cells
We next investigated which of the three known ligands of Ptch2 could be released in BTICs-CM or in endothelial cell-CM. DHH was notably detected in the 14 different BTICs-CM at the average concentration of 15 ng/ml, while sonic hedgehog (SHH) could not be detected (Figure 3A). Because endothelial cells do not secrete any of these ligands, unlike U87 glioma cells, an autocrine action of HH ligands on brain endothelial cells was discarded ( Figure 3A). Thus, we further investigated how BTICs-secreted DHH could modify endothelial homeostasis. Upon DHH silencing, BTICs-mediated Ptch2 upregulation was quelled, suggesting that Ptch2 could be its own direct target in a positive feedback loop, as demonstrated previously in HH signaling (Figures 3B,C,D; Evangelista et al., 2006;Aberger et al., 2012). Interestingly, BTICs-CM no longer induced permeability (Figure 3E), and depletion of DHH dramatically reduced the

FIGURE 3 | DHH mediates the BTICs-CM effects in brain endothelial cells. (A)
SHH and DHH secretion was examined by ELISA in BTICs and endothelial cells (EC) conditioned media. U87 were used as a positive control. (B) BTICs cells were transfected with either control (sic) or siRNA against DHH (siDHH). Conditioned media was collected and processed for DHH secretion by ELISA assay. Alternatively, mRNA were extracted from both sic and siDHH receiving cells, and DHH expression was assessed by RT-PCR. GAPDH was used as a reference gene. (C-G) Confluent monolayers of hCMEC/D3 cells were exposed for 24 h to BTICs-conditioned media depleted (siDHH) or not (sic) for DHH. Ptch2 expression was assessed by RT-PCR (C) and flow cytometry analysis (D). Dashed histograms correspond to the isotype-matched control (Ig), black and blue histograms show antibody staining in endothelial cells treated as indicated. (E) Permeability of endothelial cells was analyzed. (F,G) Endothelial cells were also tested for their sprouting (F) and tube formation (G) ability. The number of sprouting cells and mean sprout length were determined in (F). Tube length, and number of branch points were counted in (G). ***p < 0.001, **p < 0.01 by analysis of variance (ANOVA). Each panel is representative of at least two independent experiments.
BTICs-CM pro-angiogenic effects, as assessed by sprouting and tubulogenesis ability (Figures 3F,G). Taken together, these results indicate that BTICs induce the expression of Ptch2 receptor in endothelial cells, while producing DHH ligand in the medium, thus enabling a paracrine DHH/Ptch2 signaling cascade that culminates in endothelial remodeling.

Desert Hedgehog Drives Vascular Dysfunctions in Mice
Finally, we explored the contribution of the paracrine DHH/Ptch2 signaling in vivo, using ectopic subcutaneous xenografts. Importantly, DHH silencing did not influence BTICs tumorspheres formation and viability (Figures 4A-C). Indeed, DHH-silenced BTICs maintained their self-renewal ability, as evidenced by the tumorspheres formation (Figure 4B), while their viability rate remained similar to control cells ( Figure 4C). BTICs stably depleted for DHH expression were engineered and two clones (sh2 and sh4) were selected for their silencing efficiency and compared to cells that receive a non-silencing shRNA (shC). Accordingly, DHH expression and secretion were dramatically reduced in both sh2 and sh4 clones ( Figure 4D). The reduction of DHH production had no significant effect on tumor initiation in vivo, as the number of tumor-bearing mice scored higher than 2/3 in each group within 12 weeks ( Figure 4E). However, DHH loss-of-function in BTICs had a dramatic effect on tumor growth, as sh2 and sh4 clones produced tumors four times smaller than controls ( Figure 4F). Likewise, vessels (CD31 staining) and hypoxic zones (Hpi staining) density were alleviated in DHH-less xenografts ( Figure 4G). Accordingly, this effect was associated with a restrained intra-tumor vascular leakage ( Figure 4H). Collectively, these results unveil a role for DHH in exacerbated tumor angiogenesis and permeability, which can ultimately favor glioblastoma growth, and thus place the DHH/Ptch2 nexus as a potential candidate for therapeutic intervention.

DISCUSSION
In summary, we report here that glioblastoma-initiating cells, which compose a small fraction of cells able to self-renew and drive tumorigenesis, promote brain endothelial cell remodeling though aberrant HH signaling. Indeed, we found that BTICs released DHH ligand in the extracellular space, while inducing Ptch2 expression in endothelial cells. Additionally, DHH ligand elicits a signaling cascade, through its receptor Ptch2, ultimately impacting on endothelial plasticity. The HH family is composed of three members, Sonic (SHH), Desert (DHH), and Indian (IHH) that serve as ligands for PTCH receptors, Ptch1 and Ptch2 (Echelard et al., 1993;Riddle et al., 1993). Of note, the HH pathway is aberrantly activated in many cancers, and contributes both to tumor progression in various organs such as brain, lung, and skin (Vorechovsky et al., 1997;Evangelista et al., 2006) and to control the fate of tumor-initiating cells (Bar et al., 2007;Justilien et al., 2014;Takebe et al., 2015). Indeed, HH plays an essential role in driving tumorigenesis and drug resistance (Filbin et al., 2013), along with favoring cancerinitiating phenotype (Bar et al., 2007). HH can also enhance the expression of angiogenic factors, such as angiopoietins (Lee et al., 2007). Interestingly, we found that DHH does not seem to be involved in maintaining BTICs. Indeed, DHH silencing did not influence BTICs tumorspheres formation and viability. Conversely, DHH largely contribute to BTICs-CM impact on brain endothelial cell behavior in vitro and tumor angiogenesis in vivo. In keeping with this idea, it has been reported that HH nexus is instrumental in maintaining the blood-brain barrier integrity during both development and adulthood in mouse models (Alvarez et al., 2011). In this context, pharmacological inhibitors targeting Src kinase activity did not fully abrogate BTICs-CMtriggered endothelial behavior. Indeed, the partial blockade of endothelial permeability increase obtained by Src inhibition might be attributed to the presence of other pro-permeability factors in the BTICs-CM, among which are Semaphorin 3A and VEGF-A (Bian et al., 2007;Dwyer et al., 2012;Le Guelte et al., 2012;Treps et al., 2015). Meanwhile, hindering VEGF-R2 tyrosine kinase activity did alter BTICs-CM action on brain endothelial cells. Overall, these observations suggest that DHH might operate independently of VEGF-A in the tumor microenvironment. Thus, DHH might emerge as a novel pro-permeability factor and a novel candidate for anti-edema and anti-angiogenic therapeutic actions.
Whereas Ptch1 signaling is widely documented, Ptch2 modus operandi and functions continue to be elucidated. For instance, Ptch1 triggers both a canonical and non-canonical signaling pathways (Aberger et al., 2012), and can synergize with PI3K pathway to promote tumor growth and viability in GBM. Accordingly, blockade of both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis (Filbin et al., 2013). Here, we found that Ptch1 expression in endothelial cells was not affected by BTIC-CM challenge. Likewise, silencing Ptch1 fails to stop BTIC-mediated endothelial remodeling. Furthermore, SHH was not secreted by either BTICs or endothelial cells, suggesting that the canonical SHH/Ptch1 pathway does not function in the co-culture system. Interestingly, it has been recently documented that Ptch2 could suppress SHH signaling (Alfaro et al., 2014). In this scenario, Ptch2 expression in endothelial cells might counteract the effects of SHH locally secreted by other cell types from the tumor mass, such as astrocytes and stromal cells. Collectively, our data suggest that Ptch2 upregulation in endothelial cells and further ligand activation cause an imbalanced HH signaling, that can account for the observed vascular dysfunctions and barrier disruption, while sparing SHH positive impact on tumor cell expansion.
Our results demonstrate that targeting the DHH/Ptch2 nexus emerges as an appealing approach to curb vascular dysfunctions in GBM. Indeed, ablation of HH signaling may interfere with both angiogenesis and vascular leakage, and thus tumor growth. Nevertheless, further studies addressing the receptor upregulation mechanism and its elicited signaling route are required. Overall, our work unveils a role for DHH in heightened tumor angiogenesis and permeability, which may ultimately favor GBM progression, and thus places the DHH/Ptch2 nexus as a putative target for novel therapies directed against malignant stem-cell niches in GBM.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry

text:
Phosphorescence Monitoring of Hypoxic Microenvironment in Solid-Tumors to Evaluate Chemotherapeutic Effects Using the Hypoxia-Sensitive Iridium (III) Coordination Compound

Objectives To utilize phosphorescence to monitor hypoxic microenvironment in solid-tumors and investigate cancer chemotherapeutic effects in vivo. Methods A hypoxia-sensitive probe named BTP was used to monitor hypoxic microenvironment in solid-tumors. The low-dose metronomic treatment with cisplatin was used in anti-angiogenetic chemotherapeutic programs. The phosphorescence properties of BTP were detected by a spectrofluorometer. BTP cytotoxicity utilized cell necrosis and apoptosis, which were evaluated by trypan blue dye exclusion and Hoechst33342 plus propidium iodide assays. Tumor-bearing mouse models of colon adenocarcinoma were used for tumor imaging in vivo. Monitoring of the hypoxic microenvironment in tumors was performed with a Maestro 2 fluorescence imaging system. Tumor tissues in each group were harvested regularly and treated with pathological hematoxylin and eosin and immunohistochemical staining to confirm imaging results. Results BTP did not feature obvious cytotoxicity for cells, and tumor growth in low-dose metronomic cisplatin treated mice was significantly inhibited by chemotherapy. Hypoxic levels significantly increased due to cisplatin, as proven by the expression level of related proteins. Phosphorescence intensity in the tumors of mice in the cisplatin group was stronger and showed higher contrast than that in tumors of saline treated mice. Conclusions We develop a useful phosphorescence method to evaluate the chemotherapeutic effects of cisplatin. The proposed method shows potential as a phosphorescence imaging approach for evaluating chemotherapeutic effects in vivo, especially anti-angiogenesis.

Introduction
Tumor is an extremely common problem for human beings, and chemotherapy is still the main treatment measure for tumor therapy. In clinical trials, the tumor size is a main and superficial characteristic without monitoring of microenvironment. As investigators do not know more if they don't have complicated analysis, they can neither evaluate chemotherapeutic effects in perspective nor design new chemotherapy regimens to predict its effects. Therefore, a reasonable method is urgently needed for evaluating chemotherapeutic effects. To develop such a method, a specific landmark of tumor which is significantly different with normal tissues should be indicated.
Recent studies found hypoxia is a key feature in the solid-tumor microenvironment [1], facilitates tumor progression, metastatic spread, and resistance to radiation and chemotherapy. Hypoxia-inducible factor 1 (HIF-1) is the most significant hypoxia-related protein, and its cellular signal pathway has been studied for many years [2]. HIF-1 can activate transcription of many genes involved in angiogenesis, cell growth and survival, glucose metabolism, invasion, metastasis, and drug resistance, to name a few [3]. Angiogenesis-related protein vascular endothelial growth factor (VEGF) and multidrug resistance 1 (MDR1) are up-regulated by hypoxic conditions in tumor cells. Angiogenesis is mostly an adaptive response to tissue hypoxia, and also an essential requirement for neoplastic growth [4]. Hence, hypoxia and angiogenesis are hallmarks of cancer and major targets in cancer therapy, and several target-hypoxia/angiogenesis agents are being actively studied for their antitumor activity in preclinical models and/or in clinical trials [5].
The impact of chemotherapy on the tumor oxygenation environment remains controversial. Early work from pioneers, such as computed tomography (CT) perfusion [6] and dynamic contrast-enhanced magnetic resonance imaging (MRI) [7], suggest that reduction of newly formed blood vessels starves tumors from nutrients and oxygen, thereby reducing tumor growth and inducing tumor cell death (starvation hypothesis). According to the Jain's hypothesis, treatment with bevacizumab can primarily improve chemotherapeutic efficacy by normalizing the tumor vasculature, resulting in more effective oxygen and drug delivery [8]. What is clear in these tumor studies is that the mechanisms of chemotherapy are fairly complex and depend on the tumor type, hypoxia detection time point, and hypoxia modality [9]. Various cytotoxic chemotherapeutic agents have anti-angiogenesis effects [10,11], and low-dose metronomic chemotherapeutic treatment with cisplatin can affect angiogenesis [12,13]. To date, however, intuitive and direct methods for detecting changes in tumor hypoxic microenvironment resulting from chemotherapy are limited [14][15][16].
Several measures have been developed to observe and measure hypoxic microenvironment. The most accurate method involves the use of an oxygen electrode to penetrate the tumor tissue [17]; this method, however, is invasive and may only measure O 2 in tissues where penetration occurs. As well, the oxygen electrode may directly consume O 2 . Therefore, noninvasive approaches are necessary to monitor hypoxia in tumors both accurately and reliably.
A number of noninvasive imaging techniques for measuring tumor hypoxia have been exploited, including radionuclide imaging (positron emission tomography [PET] and singlephoton emission computed tomography [SPECT]), MRI, and optical imaging [18]. In hypoxia monitoring, nitroimidazole compounds, such as fluoromisonidazole (FMISO) [19] and fluoroazomycinarabinoside (FAZA) [20], have been developed as hypoxia-targeting PET probes for noninvasive hypoxia monitoring. Cu (II)-ATSM is another PET probe without nitroimidazole. These probes radiolabeled with radioisotope can be reduced by enzyme systems in hypoxic tissue, which is believed to bind to macromolecules [21][22][23]. Among these imaging techniques, optical imaging has several advantages, including high sensitivity and resolution, low imaging cost, and lack of radioisotope requirement. Phosphorescent coordination compounds show luminescent emissions that are distinct from those of the tumor hypoxia targeting probes described above. They are distributed systemically through the whole body and emit phosphorescence where the oxygen supply is insufficient.
Iridium (III) coordination compounds are superior to other phosphorescence-emitting probes, such as platinum (II)-and palladium (II)-porphyrins, and ruthenium (II) coordination compounds, because of their small size and suitable phosphorescence lifetime of the former; these characteristics are favorable for oxygen sensing in living animals [24]. Furthermore, the optical properties of iridium (III) coordination compounds may be tuned by adjusting ancillary ligands [25].
The purpose of the present study is to develop a phosphorescence imaging approach using BTP to monitor hypoxic changes resulting from chemotherapeutic treatment in vivo. We hypothesize that the hypoxia-sensitive probe BTP is useful for quantitatively evaluating changes in tumor hypoxia during chemotherapy. We designed an experiment using BTP phosphorescence imaging to monitor tumor hypoxic microenvironment in vivo during cisplatin treatment to assess chemotherapeutic effects and the reasonability of treatment (Fig. 1). The results of this paper agreed with our hypothesis; this sensor showed great potential for hypoxia-targeting cancer imaging in vitro and in vivo, and might be used as a potent biomarker for real-time monitoring of hypoxic microenvironment and detection of overall changes resulting from chemotherapeutic treatment. Therefore, BTP may be applied as a promising and novel phosphorescence imaging method for evaluating chemotherapeutic effects in vivo.

Materials and Methods Materials
BTP synthesized according to the literature was purchased from American Dye Source, Inc. Mouse monoclonal antibodies to HIF-1α and VEGF were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Other reagents were purchased from Aladdin Chemical Reagents Co., Ltd. All reagents were of analytical grade and directly used without further purification.
A mouse colon adenocarcinoma (CT-26) cell line [28] was purchased from the Chinese Academy of Sciences (Shanghai). Cell culture consumables were purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). A streptavidin-biotin2 system labeled with horseradish peroxidase (LSAB2 System-HRP) was purchased from Dako Co., Ltd. Male nude BALB/c mice were purchased from the Department of Experimental Animals, Shanghai Medical College, Fudan University. The mice were raised under a standard housing environment until 6 weeks of age and featured body weights of at least 25.0 g. All animal experiments were approved by the Ethics Committees of Xi'an Jiaotong University.

Phosphorescence properties of BTP
A spectrofluorometer (FluoroMax-4, HORIBA JobinYvon, France) was used to measure the phosphorescence spectra of BTP under different conditions (hypoxia and normoxia). Briefly, 5 mg of BTP was dissolved in 35 mL of DMSO; 3 mL of this mixture was pipetted into a quartz cuvette. Phosphorescence excitation spectroscopy was performed at an excitation wavelength of 480 nm.
To observe the phosphorescent performance of BTP in a hypoxic atmosphere, two quartz cuvettes with 3.0 mL of BTP were placed under a handheld UV lamp at an excitation wavelength of 365 nm in a dark room. Photographs of the cuvettes were obtained by a camera (Powershot A650IS, Canon, Japan). Nitrogen was used to purge the air out from one of the quartz cuvettes; the other tube was used as a control. In both tubes, the BTP concentration was set to 0.14 mgÁmL −1 .

Cytotoxicity evaluation of BTP
To evaluate the cytotoxicity of BTP, CT-26 cell necrosis and apoptosis were evaluated in cell cultures to which BTP had been added for up to 72 h using trypan blue dye exclusion and Hoechst 33342 plus propidium iodide (PI) assays. BTP concentrations of 0, 1, 5, 20, 50, 100 μM were tested. Cholesterol was used as a negative control and 7-ketocholesterol [29] was used as a positive control; both molecules feature lipophilicity similar to that of BTP [30].

Cell uptake of BTP
CT-26 cells were cultured in DMEM medium containing 25 mmolÁL −1 of glucose with 10% FBS at 37°C and then cultured under 5% or 20% O 2 in a concentration-changeable multi-gas incubator (MCO-5M, Sanyo, Japan) for 24 h at 37°C. Afterward, BTP dissolved in DMSO was added to the medium at a final concentration of 5 μM for 2 h. An inverted fluorescence microscope (Eclipse Ti-S, Nikon, Japan) with a camera was used to obtain phosphorescence images.

Nude BALB/c mice model of colon adenocarcinoma
Tumor transplants were established in male nude BALB/c mice by injection of 5×10 6 mouse colon adenocarcinoma-derived CT-26 cells to the left anterior axilla. Experiments with tumorbearing mice were performed one week after injection of tumor cells.

Low-dose metronomic chemotherapy treatment with cisplatin
To evaluate hypoxic changes resulting from chemotherapy, all mice were treated for 21 days with low-dose metronomic cisplatin, a well-studied anti-cancer chemotherapeutic drug commonly used against different human tumors. When the tumor volumes approached 60-70 mm 3 , saline (100 μL) and cisplatin (1 mgÁkg −1 , 100 μL) were intraperitoneally injected into the 12 CT-26 tumor-bearing nude mice every two days (6 with saline, 6 with cisplatin).

Monitoring tumor hypoxic microenvironment in vivo
The tumor size in each mouse was measured every three days after tumor implantation by a caliper and calculated as follows: volume = (tumor length) × (tumor width) 2 /2. Every three days, hypoxic changes in tumors in vivo were monitored by carrying out optical imaging, beginning from day nine post-treatment as described above. BTP DMSO/saline solution was injected intravenously into the animals 2 h before imaging. Phosphorescent images of the whole mouse body as well as a close-up image were obtained using an in vivo fluorescence imaging system (Maestro 2, CRI, USA) under the following conditions: "Blue" filter model; excitation filter range, 455 nm (435-480 nm); emission filter range, 490 nm longpass; acquisition setting, 500 to 720 nm in 10 nm steps. The auto-fluorescence of mice and BTP phosphorescence were obtained and then unmixed using Maestro spectral software.

Pathological and immunohistochemical studies
Tumors were quickly removed and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. After fixation, tumors were immersed in a series of sucrose solutions with increasing concentrations for 48 h at 4°C. For the pathological study, tumor samples were embedded in Tissue-Tek O. C. T. compound mounting medium (Sakura Finetek Inc., CA, USA), frozen in liquid nitrogen, and cryostat-sectioned (5 μm) by a cryostat (Jung CM3000, Leica, Germany). Histological sections from cryostat blocks were stained with hematoxylin and eosin (H&E). Tissue antigens were retrieved after formalin-fixing and paraffin-embedding, and antigens were stained with the primary antibodies (mouse monoclonal antibodies to HIF-1α or VEGF; 1:200 dilution). A second immune reaction was performed using LSAB2 System-HRP. The quantitative data of IOD SUM were extracted using Image-Pro Plus 6 (Media Cybernetics Co., Ltd., USA).

Statistical analysis
Data were presented as mean ± SE. Three repetitions or sets were performed. T-test was performed to determine differences among samples. A p value of less than 0.05 was considered significant ( Ã p<0.05, ÃÃ p<0.01, ÃÃÃ p<0.001). The Software packages of GraphPad Prism 5 (GraphPad Software, Inc., USA) and SPSS 19.0 (IBM Co., USA) were used in the statistical analysis.

Phosphorescence properties of BTP
The chemical structure of BTP is shown in Fig. 1. BTP had a broad excitation wavelength ranging from 400 to 550 nm. When excited at its maximum excitation wavelength (480 nm), BTP showed a maximum emission peak at 612 nm, with a shoulder at 667 nm ( Fig. 2A). DMSO made the spectra shift red compared with low polarity solvent. Part of the emission curve extends to the near-infrared region (650-900 nm), which is the preferred optical window for in vivo imaging because of the relatively low tissue absorption and auto-fluorescence in this region. BTP emits strong phosphorescence where the O 2 supply is insufficient. The intensity of emission peaks changed under different conditions (hypoxia and normoxia), and the peak intensity of the compound in pure nitrogen was about 3.5 times that in the atmosphere [27].
Phosphorescent images of BTP samples under normoxic (left) and hypoxic (right) conditions are shown in Fig. 2A. After pumping nitrogen through the BTP solution to generate a hypoxic environment, the phosphorescence signals increased significantly in the test quartz cuvettes. The relationship between pO2 and phosphoresce intensity are shown in Fig. 2B. According to Stern-Volmer equation, Q 0 /Q and τ 0 /τ values are proportional to pO2, and our results are similar to the report [31]. Therefore, phosphoresce intensity (Q) is inversely proportional to pO2.

Cytotoxicity evaluation of BTP
To evaluate the cytotoxicity of BTP, we examined cell viability according to necrosis and apoptosis in CT-26 cells using trypan blue dye-exclusion and Hoechst 33342 plus PI assays. Necrosis and apoptosis of cells treated with BTP were comparable with those observed in cells treated with the cholesterol control (Fig. 2C). Treatment with BTP concentrations of less than 100 μM yielded a cell necrosis rate of less than 10% and a cell apoptosis rate below 2%. In our previous discussion, we postulated that any cytotoxicity observed might be attributed to the presence of organic solvents (DMSO as the solvents of BTP) injected along with BTP. The water soluble nanoparticles containing a BTP derivative was low cytotoxicity to avoid used organic solvent. In fact, BTP does not have obvious cytotoxicity if solvent is used less than 2% in cell medium (v/v), and may be used as a safe phosphorescence probe in vivo.

Cell uptake of BTP
Tumor cell cultures were utilized to examine cell uptake of BTP. CT-26 cells were treated with 5 μM of BTP under 5% (hypoxic) or 20% (normal) O 2 and cultured for 2 h. Results showed that hypoxic CT-26 cells displayed higher phosphorescence signals than cells under normal pO 2 (Fig. 3).
Monitoring tumor hypoxic level changes in microenvironment in vivo and the pathological and immunohistochemical studies CT-26 tumor-bearing nude mice were treated with low-dose metronomic curing with cisplatin, and BTP accumulation in the tumor site was observed using a Maestro 2 fluorescence imaging system. Fig. 4A shows significant inhibition of tumor growth in cisplatin-treated mice (p<0.01) compared with that in saline control mice; BTP signals significantly increased over the treatment period (Day 18: p = 0.049; Day 21: p<0.01). This phenomenon indicates that cisplatin inhibited tumor growth and increased tumor hypoxia. Using the same imaging technique in vivo, a mouse treated with saline and another mouse treated with cisplatin were observed continually from Days 9 to 21. Fig. 5 illustrates that the mouse in the saline group showed gradual increases in tumor size as the phosphorescence intensity increased. By contrast, the mouse in the cisplatin group displayed a smaller tumor as well as a sharper increase in phosphorescence intensity. Here, we defined phosphorescence intensity using a color bar: long-wavelength colors present strong intensity while short wavelength colors mean weak intensity. Images of the groups on Day 18 (when significant differences were first observed) are shown in Fig. 6. While the tumor size in mice in the saline group was higher than that in mice in the cisplatin group, the images did not show clear boundaries and the morphology of tumors could not be examined completely. Besides the strong phosphorescence intensity of tumors in mice in the cisplatin group, a high-contrast contour might also be observed, which indicated that O 2 distribution between tumors and normal tissues was quite different, especially under chemotherapeutic conditions, when changes in the tumor microenvironment might occur.
Upon completion of tumor hypoxia imaging, the CT-26 tumor-bearing mice were sacrificed. Tumors were harvested, sectioned, subjected to H&E staining, and then immunohistochemically stained with HIF-1α or VEGF (Fig. 7). Pathological sections illustrate that tumor cells died and tissues appeared empty under chemotherapy. The immunohistochemical stain of HIF-1α shows that HIF-1α expression was higher in the cisplatin group (darker brown area, Day 9: p = 0.013; Day 21: p = 0.487) than in the saline group, which indicated a positive relationship with hypoxic conditions. The immunohistochemical stain of VEGF shows that VEGF expression was higher in the cisplatin group than in the saline group. However, it had no significant difference between two groups (Day 9: p = 0.339; Day 21: p = 0.128) for two reasons. One was the suppression of cisplatin, and the other was induced expression by HIF-1. They made VEGF neither increased nor decreased. Quantitative data of IOD SUM were extracted and shown in Fig. 4C and 4D. HIF-1α expressed a trend similar to that of phosphorescence and BTP under 5% O 2 (BTP hypoxia). There are three columns including a bright field, a dark field and a merged field. The first column presents the bright field under an inverted microscope; the second column shows the phosphorescence images of cells as the bright field graphs without any moving, and the third one merged them.   Phosphorescence Evaluate Chemotherapeutic Effects in Solid-Tumors intensity, which showed a linear relationship with O 2 concentration (Stern-Volmer equation) [32]. HIF-1α expression was always higher in the cisplatin group than that in the saline group. As an up-regulated protein by hypoxia, VEGF expression gradually decreased in the cisplatin group but initially increased and then decreased in the saline group ( Fig. 4C and 4D).

Discussion
Commonly used measurement for tumor hypoxia includes HIF-1α immunohistochemistry assessment, oxygen electrode probe, PET radiotracers such as 18 F-FMISO, 18 F-FAZA, Cu (II)-ATSM. Oxygen electrode probe usually provides a minimum invasive measurement for the local oxygen level and may vary largely by the detecting position. In contrast, PET hypoxia imaging can offer an alternative way with non-invasive, overall and accurate hypoxia imaging. However, it may require the high cost facilities and has radiation exposure. In the current study, we used HIF-1α immunohistochemistry staining to validate our findings of BTP-based imaging. However, it is beyond the scope of the current study. Further study may involve a To explain the various phenomena in our research, we focus on the HIF-1 cellular signal pathway, which has been investigated for many years [2]. HIF-1 can activate transcription of many genes involved angiogenesis, cell growth and survival, glucose metabolism, invasion, metastasis, and drug resistance, among other [3]. HIF-1α protein expression is similar in normoxic and hypoxic conditions but tends to stabilize in hypoxic environments. HIF-1α combines with HIF-1β and activates several HIF-1 target gene to express proteins, such as VEGF [33,34], which is a significant protein involved in angiogenesis. Overexpression of VEGF can contribute to so many diseases, especially solid-tumors. Tumors are unable to grow beyond a limited size in the absence of adequate blood supply. HIF-1-mediated VEGF leads to angiogenesis, resulting in remittance of hypoxic conditions and tumor metastasis. A rapid rate of tumor growth makes the blood supply again become inadequate, the new circle will occur and finally a balance will be achieved. In Fig. 8, hypoxia-influencing factors are illustrated in a chain. Factors marked with "+" indicate a positive correlation, and factors marked with "-" indicate a negative correlation. The hypoxic level kept spiraling, which was confirmed by BTP phosphorescence in our research that was shown in Fig. 4B.
When tumors are treated by chemotherapy, the hypoxic condition is changed. Cisplatin, a well-studied and commonly used anti-cancer chemotherapy drugs against different human tumors, was used in the present work as a model drug. These series of non-target drugs react in vivo, binding to and causing crosslinks of DNA, which ultimately trigger apoptosis. However, previous reports have shown that various cytotoxic chemotherapeutic agents exert anti-angiogenesis effects because the response of chemotherapeutic drugs to vascular endothelial cells were 10-10,000 times more sensitive than that of tumor cells [10,11]. Although cisplatin is not a target drug of angiogenesis, low-dose metronomic treatment with cisplatin yields anti-angiogenesis effects and restrains the expression of HIF-1α and VEGF [12,13]. If these proteins are restrained, the angiogenesis is suppressed, but the hypoxic condition may be intensified. In our Phosphorescence Evaluate Chemotherapeutic Effects in Solid-Tumors studies, low-dose metronomic treatment with cisplatin caused anti-angiogenetic effects, and less O 2 could be delivered to tumors. Finally, BTP phosphorescence intensity increased but the level of HIF-1α was influenced by both chemotherapy and pO 2 in different directions, and its expression would be relied on whose power was stronger.
Angiogenesisis one of the fastest growing fields in biomedical research, and advances in our basic understanding of the process and its clinical application have been rapidly increased. However, the effects of angiogenesis remain controversial. Angiogenesis is a complex, highly orchestrated process that plays a critical role in the normal development and pathophysiology of common diseases. The conventional rationale of anti-angiogenesis therapy is that suppression of angiogenesis and blood supply, which can cause tumor death through ''starvation" [4]. However, the concept of ''vascular normalization," which was proposed by Jain et al. [8], provides another explanation for this phenomenon. Jain et al. hypothesized that anti-angiogenesis actually ''normalizes" the tumor vasculature, transiently increases blood perfusion within the tumor, and alleviates hypoxia, thereby enhancing the delivery of oxygen and drugs to tumor cells to increase the malignancies' response to chemotherapy [35]. These phenotypic changes in vasculature represent a response to changes in the balance of pro-and anti-angiogenesis factors in the tumor tissue [8].
In this current work, we made a proposal that three cases (Type I, II and III) were in the balance of pro-and anti-angiogenesis factors (Table 1). Type I: investigations of predecessors showed that tumor cells become anoxic, inducing cell death [36]. In this case, the anti-angiogenesis affects blood vessels inadequately, causing an increase in the phosphorescence intensity with shortening tumor volumes. However, these anti-angiogenesis drugs are seldom available. Type II: if the anti-angiogenesis effects are not enough, the cells under non-fatal hypoxia may Phosphorescence Evaluate Chemotherapeutic Effects in Solid-Tumors increase the chemotherapy or radiotherapy resistance [37][38][39], and natural selections will choose the cells with higher tolerance of hypoxia [40]. In this situation, the volume of tumors is inhibited with a low growth speed but the phosphorescence intensity is increased. Type III: vascular normalization has occurred, enhancing the delivery of oxygen and drugs to tumor cells [8], and the phosphorescence intensity is down, but the tumors rapidly grow. Besides, we were unable to find that the volume of tumors decreased with low phosphorescence intensity. We predict it can be observed when tumors are cured.
According to the data of phosphorescence intensity, pO 2 in tumors could be calculated using the Stern-Volmer equation: where Q 0 , κ q , and τ 0 in tumor tissues were obtained from our current and previous work (Q 0 = 7.611×10 9 photÁcm -2 Ás -1 , κ q = 1.2×10 4 mmHgÁs -1 , τ 0 = 9.05 μs) [26,27]. The results are shown in S1 Table. The measurable range is shown in Fig. 2B, in which 0-60 mmHg is suitable to be measured with BTP; the relationship between three types in the balance of pro-and anti-angiogenesis is summarized in Table 1. The distinguishing standard of three types (pO 2 in tumors) is set by our data, oxygen dissociation curve of capillary vessels [41], and reference [42]. Other groups showed different results with different dose of treatment, which were shown in S1 Fig. This is the reason that the anti-VEGF agents were singly used, whose therapy effects might be weak and disappointing. Targeting of hypoxia in cancer therapy must consider the combination styles and doses of drugs. For example, chemotherapeutic drugs combined with anti-VEGF inhibitors may cause serious hypoxic conditions in tumors and increase the expression of HIF-1, which induces genes involved in chemotherapy resistance [37][38][39]. Normalization of the tumor vasculature may also occur since the tumor volume depends on the cytotoxicity of chemotherapeutic drugs. In our study, BTP phosphorescence indicated that low-dose metronomic therapy with cisplatin could adequately suppress angiogenesis, though we did not combine it with VEGF targeting drugs, which belonged to Type II drug effects. However, some cases in our investigation showed that different doses or combinations might cause Type III phenotypic in S1 Fig., and Type I is seldom found because hypoxia-induced cell death is difficult to achieve by chemotherapy or anti-VEGF therapy. For example, the new cisplatin group was close to Type I in the beginning, but became Type II accompanied with therapy, as well as combination group, and the single anti-VEGF group might be in Type III. The phosphorescent data obtained in this work provided quantitative information on processes associated with cancer therapy. In summary, our research proposes a new phosphorescence imaging technique for monitoring hypoxic microenvironment in tumors subjected to chemotherapy. The results of our work can help evaluate therapeutic effects and present new insights into cancer prognosis and treatment. Normal tissues [26] None Low 40-60 mmHg a " − " presents a decrease in tumor size and "+" presents an increase in tumor sizes; more "+" marks indicate rapid growth. doi:10.1371/journal.pone.0121293.t001 Phosphorescence Evaluate Chemotherapeutic Effects in Solid-Tumors


extfieldsofstudy: Biology Medicine

text:
Helminths of three species of opossums (Mammalia, Didelphidae) from Mexico

Abstract From August 2011 to November 2013, 68 opossums (8 Didelphis sp., 40 Didelphis virginiana, 15 Didelphis marsupialis, and 5 Philander opossum) were collected in 18 localities from 12 Mexican states. A total of 12,188 helminths representing 21 taxa were identified (6 trematodes, 2 cestodes, 3 acanthocephalans and 10 nematodes). Sixty-six new locality records, 9 new host records, and one species, the trematode Brachylaima didelphus, is added to the composition of the helminth fauna of the opossums in Mexico. These data, in conjunction with previous records, bring the number of taxa parasitizing the Mexican terrestrial marsupials to 41. Among these species, we recognized a group of helminths typical of didelphids in other parts of the Americas. This group is constituted by the trematode Rhopalias coronatus, the acanthocephalan Oligacanthorhynchus microcephalus and the nematodes Cruzia tentaculata, Gnathostoma turgidum, and Turgida turgida. In general, the helminth fauna of each didelphid species showed a stable taxonomic composition with respect to previously sampled sites. This situation suggests that the rate of accumulation of helminth species in the inventory of these 3 species of terrestrial marsupials in the Neotropical portion of Mexico is decreasing; however, new samplings in the Nearctic portion of this country will probably increase the richness of the helminthological inventory of this group of mammals.

Introduction
Less than 25% of the 525 species of mammals distributed in Mexico have been examined for helminth parasites (García-Prieto et al. 2012). To date, 336 nominal taxa of helminths have been recorded in mammals, 26 associated with 3 species of terrestrial opossums (Virginia opossum, Didelphis virginiana Kerr, the common opossum Didelphis marsupialis Linnaeus, and the Gray four-eyed opossum Philander opossum Linnaeus) from this country. However, the knowledge of the helminth richness associated with this host group is incomplete due to the wide distribution of these mammals in Mexico. Didelphis marsupialis occurs from Tamaulipas State and west San Luis Potosí until the Yucatán peninsula. Didelphis virginiana inhabits almost all of Mexico, except for the central Plateau and Baja California peninsula. Philander opossum occurs from south Tamaulipas State along the Gulf of Mexico coast and Chiapas State (Arcangeli-Álvarez 2010, Cervantes et al. 2010). The main objective of this work is to present new records of helminth species parasitizing these 3 species of opossums in Mexico and to compare the finding to previous records.

Materials and methods
From August 2011 to November 2013, 68 opossums (8 Didelphis sp., 40 D. virginiana, 15 D. marsupialis, and 5 P. opossum) were collected in 18 localities from 12 Mexican states (Table 1), under the collecting permit FAUT 0057 issued by the Secretaría del Medio Ambiente y Recursos Naturales (SEMARNAT), Mexico. Mammals were shot by local hunters or caught with Tomahawk traps and then killed with intraperitoneal sodium pentobarbital overdose. Opossums were dissected within the following 4 h. and all organs were examined under a stereomicroscope. Helminths were placed in Petri dishes with 0.85% saline solution. Platyhelminths and nematodes were fixed with hot 4% formalin and preserved in 70% ethanol; acanthocephalans were chilled in distilled water for 10-12 h. Once the proboscis was everted, they were preserved in 70% ethanol. Platyhelminths and acanthocephalans were stained with Mayer's paracarmin, cleared with methyl salicilate, and mounted in Canada balsam. Nematodes were cleared using Amman's lactophenol and temporarily mounted for morphological study (Lamothe-Argumedo 1997). Voucher specimens of all helminth species were deposited at Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City.

Results
A total of 12,188 helminths representing 21 taxa were identified in the 68 opossums collected from 18 localities within 12 states of Mexico ( Figure 1). Six trematode, 2 cestode, 3 acanthocephalan, and 10 nematode species were collected. Below, we present Remarks. These specimens belong to A. caudalitestis due to the position of the reproductive organs and the separation of the vitelline glands in two fields lying anterior and posterior to the ovary. Furthermore, the uterus has a zig-zag shape, occupying intercecal extension and the S-shape of the excretory vesicle, sinuous between both testes (Caballero et al. 1952).

Brachylaima didelphus Premvati & Bair, 1979 ‡
Site of infection. Intestine.  Remarks. The specific identification of this material follows Premvati and Blair (1979) and is based on the disposition of the vitellaria which extending from pharynx to posterior end.
Remarks. In accordance with Thatcher (1970) this species is characterised by having body flattened and pyriform, covered with small spines. Cirrus and cirrus sac large. Parasites in gall-bladder of marsupials.
Remarks. Rhopalias caballeroi is distinguished by the absence of oral and flanking spines, and because it has between 4 and 11 spines visible within tentacle sacs (Haverkost and Gardner 2008). ( Remarks. The diagnostic traits of this species are: flanking and oral spines present. Between 3 and 11 spines visible within tentacle sacs, which extend far beyond the posterior margin of the pharynx (Haverkost and Gardner 2008). Remarks. This species was identified by having tentacle sacs that do not extend beyond the posterior margin of the pharynx and by having only flanking spines (Haverkost and Gardner 2008 Remarks. In accordance with Cañeda-Guzmán et al. (2001), T. didelphidis is distinguished by the morphology of the scolex that is formed by 4 well separated lobes each containing 1 noncircular sucker opening laterally inside the exterolateral cavity, a large-sized body and by the shape of gravid proglottids that are inversely craspedote, among others.
Oncicola luehei (Travassos, 1917 Remarks. These specimens belong to O. luehei because the dimensions of the proboscis, the number of hooks (36), as well as its size and arrangement fits to the morphology mentioned by Machado (1950). Remarks. Our material was identified following Prestwood (1976); this species is characterised because the oral opening is surrounded by lips, the morphology and size of the spicules and the number and arrangement of bursal rays.

Family Aspidoderidae Skrjabin & Schikhobalova, 1947
Aspidodera raillieti Travassos, 1913 Site of infection. Intestine. Remarks. We identify these nematodes according to the re-description made by Adnet et al. (2009), who established the number of caudal papillae (ten pairs of button-like papillae, symmetrically ventro-laterally located), as well as the single median papilla at the anterior cloacal lip and four pairs of post-cloacal papillae, as diagnostic traits of this species.
Remarks. These specimens were identified based on the re-description of this species (Matey et al. 2001). Its diagnostic traits are: the presence of 2 spongelike areas on the inner side of each pseudolabia, and the number of caudal papillae (22).

Family Trichuridae Railliet, 1915
Trichuris didelphis Babero, 1960 Site of infection. Caecum. Remarks. Our material was identified based on the original description (Babero 1960). This species is characterised by the size of the spicule (0.47-0.6 mm), by having a spiny sheath, by the size of mature eggs (0.068 × 0.032 mm) and the posterior position of the vulva. Remarks. Our specimens were identified following Guerrero (1985). The synlophe of Viannaia viannai at mid-body has 3 ventral ridges orientated to left, short spicules (0.133-0.141 mm) and bursal ray arrangement 2-1-2 type.

Remarks.
To date, 12 species of the genus Travassostrongylus have been described, all parasitizing New World marsupials; Travassostrongylus orloffi Travassos, 1935 is the only species of this genus recorded in Mexico as parasite of Didelphis marsupialis; however, the finding of only 8 females make species identification difficult, because taxonomy of this group is based on male characteristics (Scheibel et al. 2014).

Discussion
As a result of this study, we reported 66 new locality records, 9 new host records, and added one species to the composition of the helminth fauna of the opossums in Mexico: the trematode B. didelphus parasitizing D. virginiana, which had not been recorded in this country (see García-Prieto et al. 2012). A total of 21 helminth taxa were obtained from the 3 opossums species analyzed (6 trematodes, 2 cestodes, 10 nematodes and 3 acanthocephalans), all in adult stage, with exception of the larvae of G. turgidum collected during their migration through the liver of the hosts. The richest helminth fauna among the 3 host species was recorded in D. virginiana, (parasitized by 17 species), followed by D. marsupialis (11 species) and P. opossum (8 species). The digestive tract had the highest number of helminth species (12 intestinals, 2 in gall-bladder, 2 in caeca, and 3 in stomach); only 2 of the 21 taxa, D. hayesi and Capillariinae gen. sp. were found in another site of infection (lungs). The geographic distribution of the helminth species was heterogeneous. The nematode C. tentaculata was the only species found in all localities. Other helminth species were collected from 7 (T. didelphis), 8 (R. coronatus) and 9 (V. viannai) localities; however, most taxa (12) were found in only one locality.
These data bring the number of taxa parasitizing D. virginiana, D. marsupialis, and P. opossum to 37, 21 and 20, respectively (García-Prieto et al. 2012). In this work we sampled in 9 previously unstudied localities; nevertheless, 47.2%, 52.4% and 40% of the taxa collected were reported previously from the Virginia opossum, Black-eared opossum and Gray four-eyed opossum, respectively. These species are typical of didelphids in other parts of the Americas (see Alden 1995;Corrêa Gomes et al. 2003;Haverskot and Gardner 2008;Bertoni-Ruiz et al. 2011;Richardson et al. 2014), conforming a group basically represented by the trematode R. coronatus, the acanthocephalan O. microcephalus and the nematodes C. tentaculata, G. turgidum, and T. turgida; these species have been recorded associated to any of the three opossum species in 7,10,15, 9, and 17 Mexican states, respectively. In states where the 3 host species are distributed sympatrically, O. microcephalus and R. coronatus are the species more frequently shared between them. On the other hand, the most restricted geographic areas are presented by the trematodes A. caudalitestis, B. didelphus, and P. magnacirrus, the acanthocephalan O. luehei, and the nematodes Gongylonema sp., and Travassostrongylus sp., which are present exclusively in one locality. In total, the records of this group of mammals come from 20 of the 32 states of the Mexican Republic; however, the geographic information is asymmetrical, because most of the samplings were made in the state of Veracruz (13 sites). Other states, as Campeche and Quintana Roo, have been sampled once. Moreover, most of the species that have been found parasitizing these didelphid species represent point locality records in only one study about its parasites cover states or regions, particularly Los Tuxtlas, Veracruz. However, the host's collections were made along 13 years, in different year season and with a very distinct sample size (see Cañeda-Guzmán 1997).
Considering only the 27 nominal helminth species recorded to date, the 3 host species shared 12 worm species along the sampled sites in Mexico; 8 were exclusively found in D. virginiana, and 2 are specialist to P. opossum. The cestode T. didelphidis and the acanthocephalan O. luehei are shared by the 2 species of the genus Didelphis but not by P. opossum; the Virginia opossum and the Grey four-eyed opossum shared the digenean D. proloba and the nematode A. raillieti, whereas D. marsupialis and P. opossum shared only P. magnacirrus. The helminth fauna of these hosts throughout their range is composed by one group of 20 specialist species, and by P. mexicanus, O. microcephalus, O. luehei, P. gethi, A. raillieti, D. longispiculata, and T. minuta that act as generalist species. Accidental species have not been reported in any of the samples carried out to date in Mexico. At a local scale, both phenomena had been also observed in marsupials of French Guiana (Jiménez et al. 2011;Byles et al. 2013).
The structuring factor of the helminth fauna in the three didelphid species is the diet; most of the helminth species infect these host species through ingestion of eggs, larvae or intermediate hosts. Fifteen of the 27 named helminth species have indirect patterns of transmission (T. didelphidis, B. didelphus, B. virginiana, D. proloba, A. caudalitestis, P. mexicanus, O. microcephalus, P. gethi, O. luehei, P. nickoli, G. turgidum, T. turgida, G. mexicanum, D. longispiculata, and D. hayesi), five are transmitted directly by eggs ingestion (A. raillieti, C. americana, C. tentaculata, T. didelphis, T. minuta) and for P. magnacirrus, R. baculifer, R. coronatus, R. macracanthus, R. caballeroi, V. didelphis and V. viannai, the life cycle is unknown (Table 2). This result is in agreement with the generalist lifestyles and diets of the three species of opossums (Krause and Krause 2006), that exposed them to the same parasite species; local differences in composition and abundance of helminth species could be related to local availability of parasites (or their intermediate hosts), as well as to the compatibility among host and helminth species, as has been showed by Cañeda-Guzmán (1997) and Jiménez et al. (2011).
The data obtained in this study came from 68 opossums collected from 18 localities (nine not previously sampled for helminths); however, the helminth fauna of each didelphid species showed a stable taxonomic composition with respect to previously sampled sites. Only one species of trematode not previously found in this group of hosts in the country was added to their parasitological record as results of our samples. In spite of the reduced scope of our samplings, this situation suggests that the rate of accumulation of helminth species in the inventory of the 3 species of terrestrial marsupials distributed in the Neotropical portion of Mexico included in this study is decreasing; however, new samplings in the Nearctic portion of this country will probably increase the richness of the helminthological inventory of this group of mammals.