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Oncolytic adenoviruses can be engineered for better tumor selectivity, gene delivery and be armed for imaging and concentrating radionuclides into tumors for synergistic oncolysis. We constructed Ad5/3-hTERT-hNIS where replication is controlled by hTERT-promoter. Ad5/3-hTERT-hNIS expresses hNIS for imaging of transgene expression and for treatment of infected tumors by radioiodine. Ad5/3-hTERT-hNIS efficiently killed prostate cancer cells and induced iodine uptake in vitro and in vivo after intratumoral virus administration. Survival of mice treated with intravenous Ad5/3-hTERT-hNIS significantly prolonged survival over mock or radioiodine only but the combination of virus with radioiodine was not more effective than virus alone. Temporal and spatial changes in hNIS-expression during therapy were detected with SPECT, demonstrating feasibility of evaluation of the combination therapy with hNIS-expressing adenoviruses and radioiodide.
Citation: Rajecki M, Sarparanta M, Hakkarainen T, Tenhunen M, Diaconu I, Kuhmonen V, et al. (2012) SPECT/CT Imaging of hNIS -Expression after Intravenous Delivery of an Oncolytic Adenovirus and 131I. PLoS ONE 7(3): e32871. https://doi.org/10.1371/journal.pone.0032871
Editor: Ilya Ulasov, University of Chicago, United States of America
Received: September 1, 2011; Accepted: February 1, 2012; Published: March 7, 2012
Copyright: © 2012 Rajecki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by the Helsinki Biomedical Graduate School (www.hbgs.helsinki.fi) and grants from Finnish Foundation for Research on Viral Diseases (www.virustauti.fi), Emil Aaltonen foundation (www.emilaaltonen.fi), Biomedicum Helsinki Foundation (www.biomedicum.fi), Irja Karvonen's Cancer Foundation (www.karvonensaatio.fi), Jalmari and Rauha Ahokas Foundation (www.ahokkaansaatio.org), The Finnish Medical Foundation (www.laaketieteensaatio.fi), K. Albin Johansson Foundation (www.foundationweb.net/johansson/), Jenny and Antti Wihuri Foundation (www.wihurinrahasto.fi), Drug Discovery Graduate School (ddgs.utu.fi), European Research Council (erc.europa.eu), ASCO Foundation (www.asco.org), HUCH Research Funds (EVO), Sigrid Juselius Foundation (www.sigridjuselius.fi/foundation), Academy of Finland (www.aka.fi), Biocentrum Helsinki (www.helsinki.fi/biocentrum), Biocenter Finland (www.biocenter.fi) and University of Helsinki (www.helsinki.fi). Akseli Hemminki is K. Albin Johansson Research Professor of the Foundation for the Finnish Cancer Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: A. Hemminki is a co-founder and shareholder in Oncos Therapeutics ltd. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.
Gene therapy with oncolytic viruses is an emerging experimental option for treating refractory cancer lacking curative therapies. This is the case for castration resistant prostate cancer that killed estimated 27,000 men in 2009 in the U.S (http://www.cancer.gov/cancertopics/types/prostate). Oncolytic adenoviruses are promising therapeutic tools since the infection results in cytolysis of transduced cells and in the release of therapeutic progeny for further spread into target tumors. The feasibility of genetic alteration of the adenoviral genome has led to the development of several modifications that render these agents cancer selective [1], [2]. Consequently, oncolytic adenoviruses have been successfully used in clinical trials, including prostate cancer patients [3]. However, although proven safe, the efficacy of the treatments still needs to be improved. One means to achieve this is to combine them with chemotherapeutics and radiation [4], [5], [6].
Human sodium/iodide symporter (hNIS) is a transmembrane protein that is located on the basolateral membrane of thyroid cells and actively transports iodine (I−) for thyroid hormone synthesis. The ability to accumulate I− via hNIS has provided the basis for diagnostic scintigraphic imaging in thyroid cancer and has been an effective means to target radioiodine for its treatment. The structure of oncolytic adenoviruses is compatible with the insertion of therapeutic molecules as transgenes and this feature has also been exploited for delivery of hNIS [7], [8], [9]. Recently, we and others have successfully used adenovirus-encoded hNIS to transport 131I into cancer cells for a combined antitumoral effect [7], [10]. In addition, hNIS-expression not only provides a therapeutic means to improve efficacy but also might serve as an imaging tool for assessing tumor size and provides information on viral kinetics during the treatment.
Recent developments in dedicated small animal multi-detector and multi-pinhole collimator scanners for single photon emission computed tomography (SPECT) have rendered the modality a powerful tool in preclinical imaging. Decades of clinical use have resulted in many commercially available tracers and kit-formulations for facile tracer radiolabeling easing the adaptation of the technique also to preclinical use. SPECT has been successfully employed in the visualization of various transgenes [11], but its advantages over other imaging modalities have rarely been more pronounced than for hNIS-imaging, where selective, readily available and inexpensive probes, 131I−, 123I− and 99mTcO4−, can be employed [10].
Telomeres are structures at the ends of chromosomes consisting of hundreds to thousands of TTAGGG-repeats and associated proteins [12]. During each mitotic cell division several telomeric repeats are lost. However, some cell types, including stem and cancer cells, maintain the capacity of indefinite renewal by expressing telomerase [13], a ribonucleotide protein that can synthesize telomeres de novo. In fact, genetic mutations leading to constitutively active telomerase-expression have been associated with most human cancers [14]. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of human telomerase and is the major determinant of telomerase activity [15]. Since hTERT is mostly active in tumor cells, or in stem cells (not effectively killed by oncolytic adenoviruses) [16], hTERT-activity can be exploited for cancer gene therapy. This approach has been successfully used in the context of genetically modified adenoviruses to obtain tumor-specific replication not only in preclinical models [17], [18] but also in one clinical trial [19].
Gene expression by oncolytic adenoviruses is determined in part by their capability to enter target cells [20]. However, the native receptor for Ad5, coxsackie and adenovirus receptor (CAR), is often poorly expressed on cancer cells [1]. Alternative targeting strategies have been developed to overcome this and several genetic modifications of the adenoviral capsid and knob have resulted in better tumor transduction [21]. One approach includes the replacement of Ad5 knob with the serotype 3 knob since, as compared to CAR, the Ad3 receptor is expressed to a higher degree on cancer tissue and is different from CAR [22], [23], [24].
In the present study, we constructed Ad5/3-hTERT-hNIS where viral replication is controlled by the hTERT-promoter. Ad5/3-hTERT-hNIS is targeted to the Ad3 receptor for effective tumor transduction and expresses hNIS in replication-permissive cells only, thus allowing imaging of viral replication and treatment of infected tumors by radioactive iodine.
Ad5/3-hTERT-hNIS expresses functional hNIS and is able to mediate iodine uptake into prostate cancer cells
Two new oncolytic adenoviruses, Ad5/3-hTERT-hNIS and its control virus Ad5/3-hTERT- Δgp19K, were constructed to study their antitumor effect against castration resistant prostate cancer. The Ad5 knob was replaced by the Ad3 knob to increase transduction of cancer cells [22]. The native E1A promoter was replaced by an hTERT-promoter for tumor-specific replication. In Ad5/3-hTERT-hNIS, the gp19K and 6.7K in E3 is replaced by hNIS that remains under the native E3-promoter and results in replication-coupled transgene expression that starts circa 8 h post-infection [25]. The control virus harbors the same deletion (gp19K and 6.7K) without insertions.
To test the capability of the viruses to induce the expression of hNIS, castration resistant prostate cancer cells were infected with the new viruses and the cells were collected for hNIS RT-PCR at 24 h or 48 h afterwards (Figure 1a). Ad5/3-hTERT-hNIS displayed strong induction of hNIS at both time points whereas the control virus -infected cells remained negative for hNIS-expression.
(a) Cells were infected with 10 vp of Ad5/3-hTERT-hNIS (lanes 1 and 3) or control virus Ad5/3-hTERT-Δgp19K (lanes 2 and 4). hNIS-RNA -expression was assayed 24 h (lanes 1 and 2) and 48 h (lanes 3 and 4) later by RT-PCR. ß-actin served as an internal control. (b) 125I uptake in prostate cancer cells infected in triplicates with 10 vp of Ad5/3-hTERT-hNIS or Ad5/3-hTERT-Δgp19K. The capability of the cells to concentrate iodide was assessed at 24 h and 48 h after infection. Student's t-test was used for statistical analyses, *p<0.05, **p<0.01, ***p<0.001 as compared to uninfected cells. Bars represent SD.
Since mRNA-expression does not automatically mean production of functional hNIS, the ability of infected cells to transport and concentrate iodine was evaluated. Ad5/3-hTERT-hNIS-infected prostate cancer cells were incubated with 125I and then analyzed for iodide uptake using a gamma-counter (Figure 1b). 24 h after infection, iodide uptake was significantly higher in Ad5/3-hTERT-hNIS infected than in mock treated cells, p<0.001 for 22Rv1 and PC-3MM2 and p = 0.003 and 0.002 for PC-3 and DU-145 cells, respectively. Significant differences were sustained at 48 h (p<0.001 for PC-3 and 22Rv1 and 0.04 and 0.003 for DU-145 and PC-3MM2 cells, respectively). PC-3 cells seemed to accumulate some radioiodine also without virus treatment. The phenomenon has also been reported previously for other cancer types and it is known that many secretory tissues, such as the prostate, feature a weak capacity to actively transport iodide [26], [27]. This suggests that possibly hNIS-independent uptake of iodine impacted the results of the in vitro study.
Ad5/3-hTERT-hNIS mediates effective oncolysis of prostate cancer cells in vitro
To determine the oncolytic capacity of the new viruses, prostate cancer cell lines were infected with viruses and cell viability was assessed by the 3-(4,5,dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)- 2-(4-sulfophenyl)-2H –tetrazolium inner salt (MTS) assay (Figure 2). Here, cell survival after infection is followed daily and the assay is stopped when the fastest virus has caused almost complete cell killing at the highest dose. In most cell lines, Ad5/3-hTERT-hNIS was more potent than wild-type adenovirus. However, some variation in the killing of different cell lines was observed, as also previously reported [24].
Prostate cancer cells were infected in triplicates with 0.01 to 100 vp/cell and the cell viability was assessed by MTS-assay. Ad5/3Luc1 is a replication-deficient adenovirus. Ad5 WT is the serotype 5 wild-type adenovirus. Ad5/3-hTERT-Δgp19K is similar to Ad5/3-hTERT-hNIS but does not contain hNIS. ***p<0.001 as compared to replication-deficient adenovirus. Bars represent SD.
In the majority of the cell lines, the transgene-less Ad5/3-hTERT-Δgp19K was more efficient in prostate cancer cell killing than Ad5/3-hTERT-hNIS (Figure 2). This may be explained by the larger genome size of Ad5/3-hTERT-hNIS, caused by the transgene, which has been reported to affect oncolytic potency [7], [10].
Iodine uptake into tumors in vivo
To evaluate the ability of Ad5/3-hTERT-hNIS to direct iodine uptake into prostate tumors in vivo, a subcutaneous tumor model was established. Each mouse had four PC-3MM2 tumors (Figure 3a). Two lowermost tumors were injected twice with Ad5/3-hTERT-hNIS and others with Ad5/3-hTERT-Δgp19K (upper right tumor) or mock (upper left tumor). To follow the kinetics of iodine accumulation into these tumors 24 h later, mice received intravenous 123I followed by a series of gamma-camera imaging between 0.5 h and 13 h after iodine administration. As seen in Figure 3a, Ad5/3-hTERT-hNIS allows in vivo accumulation of systemic radioiodine into tumors. Iodine concentration reached a plateau at 2 h post iodine-injection and remained steady until the last imaging at 13 h (Figure 3a). The variation in signal between the Ad5/3-hTERT-hNIS injected tumors is likely due to difference in tumor size as smaller tumors might have fewer blood vessels (hampering iodine access to the tumor) and may not contain as many tumor cells that allow for hNIS-expression.
(a) Tumor uptake of 123I− 0.5 h, 2 h and 13 h after i.v. administration of 123I−. The tumors were injected twice with hTERT-viruses 24 h and 48 h prior to radioiodide. 1, Mock-injected tumor; 2, Ad5/3-hTERT-Δgp19K-injected tumor; 3 and 4, Ad5/3-hTERT-hNIS-injected tumors. (b) Ad5/3-hTERT-hNIS significantly prolongs the survival of mice bearing intra pulmonary PC-3MM2 tumors. Mice received 5×1010 vp of Ad5/3-hTERT-hNIS or diluent intravenously. Next day, the mice were injected intraperitoneally with 131I−. The treatments were repeated once a week for a total of three weeks. Pairwise comparisons with the logrank test were used to compare survival curves, **p<0.01, ***p<0.001 as compared to mock-treated mice. (c) Biodistribution of 131I− in mice 48 h after the first intravenous Ad5/3-hTERT-hNIS-injection and 24 h after first radioiodide-injection. Bars represent SD.
123I accumulation was detected in the thyroid and stomach due to endogenous expression of NIS in these organs. Bladder visualization occurs due to excretion of 123I into urine [28]. Due to animal regulations, the experiment was ended at 13 h and did not permit us to study the effect of the treatment on tumor size.
Efficacy of Ad5/3-hTERT-hNIS in vivo
To study the antitumor efficacy of intravenously administered Ad5/3-hTERT-hNIS with 131I in vivo, a survival experiment was set up. PC-3MM2 cells were injected into the left lung of nude/NMRI male mice (n = 12 mice/group) to mimic aggressive, castration resistant, lung-metastatic prostate cancer. A week after cell injection, the mice received 5×1010 vp of Ad5/3-hTERT-hNIS or diluent intravenously. The next day, mice were administered 37 MBq of therapeutic radionuclide 131I or PBS intraperitoneally. This treatment-cycle was repeated once a week for a total of three weeks.
The median survival in the mock group was 19.5 days (Figure 3b). Survival prolongation was seen in the Ad5/3-hTERT-hNIS+131I group where the median survival was 28 days. This was confirmed by statistical analyses where the p-value between mock and Ad5/3-hTERT-hNIS+131I was <0.001 and between 131I only and Ad5/3-hTERT-hNIS+131I it was 0.007. As expected, 131I only (20.5 days) did not increase survival. Although intravenous delivery of oncolytic adenovirus presents many challenges, and the model used is particularly aggressive, the overall survival of virus-only treated mice was improved over mock treatment (p = 0.04). Also, one mouse was tumor-free and healthy at the end of the experiment. Survival in the virus +131I group was not improved over virus –only treatment.
As radioidide might also accumulate into organs passively, or as a result of non-tumor-specific hNIS-expression, we measured the radioactivity of different organs - including tumors - from three mice from the Ad5/3-hTERT-hNIS+131I group, killed 24 hours after the first iodide injection (48 h after first virus injection). Due to the endogenous expression of NIS in the thyroid and stomach, about 320% and 15% of the injected dose per gram of tissue (ID/g) was concentrated in these organs, respectively. Iodide uptake into tumor was 1.3% of the injected dose (Figure 3c). The high ID/g of thyroid reflects the uneven distribution of iodine: if iodine would accumulate evenly into each organ, the ID/g would be 100%/organ.
In vivo imaging of hNIS-expression by SPECT/CT after systemic administration of Ad5/3-hTERT-hNIS
While there was a survival improvement in both virus groups over no treatment or iodine only, radioiodide did not improve survival significantly. Therefore, we performed imaging-experiments to investigate reasons for this. This permitted us also to assess the feasibility of hNIS-mediated imaging. A similar setting to the survival-experiment was employed, where mice received either vehicle, Ad5/3-hTERT-hNIS only or Ad5/3-hTERT-hNIS+131I. In addition, each group received 24 h after each virus/vehicle administration an intravenous bolus of 123I− for imaging, followed by a SPECT/CT-scan and quantitation of the 123I− signal. After the first injection, 123I− uptake in the lungs was low and no clear tumor uptake was observed in mice treated with Ad5/3-hTERT-hNIS ± 131I. At this early point, 123I− uptake to the tumors was not distinguishable from the background activity, which remained at 0.00138±0.0006%ID/mm3 throughout the study (Figure 4a). This result is in accordance with the observed low tumoral iodine uptake in the biodistribution assay (Figure 3c). After the second viral injection, 3/5 of the virus-treated animals showed increased 123I− uptake in tumors, however the level of 123I− accumulation was modest. The clearest 123I− accumulation was reached after the third virus-injection, reaching at highest 0.0035%ID/mm3 (Figures 4a and 5a, c). There was no difference in tumoral 123I− accumulation between the two Ad5/3-hTERT-hNIS treated groups, regardless if they received also the therapeutic isotope 131I in addition to the imaging isotope (Figure 4a). In comparison to mice that were not treated with the virus, the difference in tumor signal was clear (Figure 5). The 123I− uptake-results are summarized in Table 1.
Mice were given Ad5/3-hTERT-hNIS or diluent and 123I− intravenously for SPECT-scanning. After the SPECT/CT-scan, mice received 131I− or PBS intraperitoneally. The iodide uptakes were calculated weekly and normalized to the injected dose of 123I−. (a) Individual tumor uptake of 123I− (the imaging isotope) in Ad5/3-hTERT-hNIS and Ad5/3-hTERT-hNIS+131I− -treated mice. All mice received 123I− for evaluation of iodide uptake by SPECT. Mice in the latter group also received 131I− right after the SPECT imaging for therapeutic purposes. Solid grey line, background level in the absence of Ad5/3-hTERT-hNIS; dotted grey lines, ± SD of background. (b) 123I− uptake in the thyroids. Results are expressed as %ID/mm3×1000 ± SD.
(a) SPECT/CT image (sagittal section) showing 123I− accumulation into tumor in the lungs of a mouse treated intravenously with Ad5/3-hTERT-hNIS and intraperitoneally with 131I−. (b) Control mouse treated with 131I− only shows no 123I− -accumulation in the lungs on the course of the treatment. (c) Transverse section of the lungs of the mouse shown in (a) shows accumulation of 123I− into the tumor on the third treatment week. (d) Transverse section of lungs of the mouse shown in (b) shows no accumulation of 123I− into the tumor on the third treatment week. The crossing point of red lines indicates tumor localization.
These data suggest that intravenous injections of Ad5/3-hTERT-hNIS allow tumor transduction, virus replication, hNIS-expression and radioiodide accumulation to such degree that it can be detected with SPECT/CT. The signal volume detected by SPECT was rather small and the mice nevertheless died, which led us to perform autopsies at the end of the therapeutic experiment. There was a discrepancy between actual tumor size seen in autopsy and the radioiodide accumulation dependent SPECT signal: tumors seemed larger than what was observed by SPECT signal. Possible explanations for this include incomplete penetration of the tumor by either virus or by 123I−.
All animals showed normal endogenous NIS expression in the thyroid, stomach, salivary and lachrymal glands. With the exception of the thyroid in 131I-treated animals, iodide accumulation into tissues expressing endogenous NIS was not affected. As the thyroid was not protected from radioiodide uptake in this experimental design, animals treated with 131I showed diminished thyroid activity in the second and third SPECT scan, as expected (Figure 4b). In addition, some mice showed physiological 123I− uptake in the esophagus, which was easily differentiated from the tumoral uptake using the anatomical information of the co-registered CT scan (Video S1).
Dose ratios (approximated as uptake ratios) thyroid∶blood∶kidney∶tumor were approximately 1000∶1.4∶1.0∶0.8 (Figure 3c). If the thyroid is protected or thyroid ablation accepted, the dose limiting normal tissue would be the red bone marrow. Assuming that the blood dose can be used as a surrogate for the bone marrow dose (i.e., assuming no active iodine trapping from blood to bone marrow and cross-dose omitted), the typical highest allowed blood dose (2 Gy/treatment cycle) would contribute 1.1 Gy to the tumor which is perhaps unlikely to result in therapeutic effect. These figures can be compared with the standard human kinetics applied for the dosimetry of internal radiopharmaceuticals (sodium iodide, oral administration, [29]) assuming 25% uptake of iodine in the thyroid at 24 h. These calculations result in thyroid∶bone marrow∶kidney ratios of 1000∶2.4∶1.05, respectively, which are in the same range as the mouse figures reported here.
After intravenous adenovirus injections, a large proportion of the dose is taken up by the liver [30]. Therefore, we analyzed the hepatic radioiodide signal which would reflect "leaky" expression of virally-encoded hNIS in a non-target organ, keeping in mind that our virus-design predicts hNIS-expression mostly in cells that allow virus replication. Only two out of five virus-treated animals showed any degree of liver accumulation of 123I− after the first virus-treatment. This is in dramatic contrast to non-regulated expression of transgenes following intravenous adenovirus injection [31]. After subsequent virus injections during the next two weeks, no iodide uptake was detected (Figure S1) and the signal difference compared to non-virus treated mice was insignificant (p = 0.24). This demonstrated that hNIS-expression from Ad5/3-hTERT-hNIS was mostly restricted to tumor cells and leaky expression in normal tissues was minimal (Video S1).
Several tissue-specific promoters have been exploited for targeting oncolytic adenoviruses and restricting their gene expression to target tumors [32]. While stringent tissue-specificity has been reported, the drawback of this approach lies in the selectivity of replication in only one or a few tumor types. However, hTERT-expression is ubiquitous in most human tumors while limited in normal somatic cells [14], making it an attractive promoter for specific adenoviral replication in human tumors. Importantly, good safety data has been obtained in a human trial featuring an hTERT-controlled oncolytic adenovirus [19]. hTERT is expressed in stem cells. However, since stem cell toxicity is not an established feature of wild type adenovirus infection [33], we can assume that stem cells have other ways to protect themselves against virus as suggested by preliminary experiments performed in normal breast stem cells [16]. Although this study used Ad5/3-hTERT-hNIS to treat castration resistant prostate cancer, Ad5/3-hTERT-hNIS could also be used to kill hTERT-positive cancer cells of different origins.
The rationale for using hNIS in cancer gene therapy has already been established [7], [8], [9], [34]. In our virus, replication-induced hNIS–expression from the adenoviral E3-region couples radiographic imaging via hNIS to viral replication kinetics. We found it feasible to assess these aspects by using SPECT. Moreover, hNIS-expression by Ad5/3-hTERT-hNIS could allow dual-modality destruction of infected tumors: therapeutic radioiodide-treatment and oncolysis. While the same cell does not need to be killed twice, the biggest utility might come from the two different bystander effects. Oncolysis can amplify the input virus dose and in theory the process continues as long as permissive cells exist. However, tumors are complex, heterogeneous and contain areas impassable for the virus, including stromal and necrotic areas. Radiation, however, could exert effects on, over and through such areas. Also, some cells are or become resistant to radiation or oncolysis [35], [36]. In this context, the strong synergy between oncolytic adenoviruses and radiation could be useful [4], [37] as suggested in previous reports [7], [9], [38], [39]. However, since there was no significant difference to the virus only in our experiment, construct design issues may play a role in the relative impact of oncolysis versus iodine mediated killing. In the context of optimized viral replication, a large sample size or an immune competent organism (where the efficacy of oncolysis could be less prominent) may be needed for demonstrating if there is any benefit of the combination in terms of efficacy.
Autopsies performed at the end of imaging experiment suggested that tumor masses were larger than what were observed by SPECT-imaging. Thus, the virus might not have been simultaneously replicating in the entire tumor and only the part of the tumor that sustains active replication at the time of imaging would be positive for hNIS-expression – uninfected or dead tissue would not be detected. The heterogeneity of the tumor mass might also contribute: although hTERT-activation would be detected in the parental tumor cells, the rapidly growing tumor bulk could allow for variation in hTERT-promoter activity, and thus in hNIS expression, due to mutations and inactivations of regulatory factors like the E-box motifs, which have been suggested to be the major determinants of hTERT-expression [40], [41]. Furthermore, intratumoral barriers including stromal, necrotic, hyperbaric and hypoxic areas are likely to hamper the penetration of the virus to the entire tumor resulting in an uneven distribution of hNIS-expression. Incomplete tumor transduction due to polarized expression of viral receptors could also play a role [42]. Finally, as predicted by the mechanism of the virus, only tumor areas through which the virus replication front is proceeding would be expected to be positive for hNIS. Areas in which virus replication has already occurred, would not be hNIS-positive, as cells that allowed for replication would have been lysed. Alternatively, oncolytic treatment might render tumor vasculature leaky or create local hypobaric areas resulting in areas of hNIS-independent iodine influx. This possibility could not be excluded since treatment with control virus was not assessed in the systemic approach. Also, in some tumors, only a small proportion of cells are malignant [43], [44], while the rest are murine-derived stromal (not permissive for adenovirus replication) and the latter would remain after oncolysis.
Visualization of the thyroid confirmed the utility of SPECT imaging in the context of radioiodide therapy. Notably, all mice treated with 131I showed diminished 123I− uptake by the thyroid during 2nd and 3rd week of the treatment. There are, however, differences regarding hNIS-expression as a transgene or as a regulator of the endocrine system. Importantly, extrathyroidal tissues expressing hNIS are unable to organify iodide and their hNIS-expression is not regulated by thyroid stimulating hormone (TSH) [45]. In contrast, hNIS-expression in the thyroid is directly regulated by serum iodine concentration and TSH [46]. TSH expression and secretion, in turn, are regulated by thyroxin and triiodothyronine, and their relationship is inversely correlated [47]. Thus, the thyroid could be protected from 131I either by iodine- or thyroxin-supplementation prior to therapy. The latter has been successfully used in glioma treatment with a lentiviral vector expressing hNIS [48]. The study reported a lack of thyroidal NIS-expression and function after a thyroxin supplemented diet whereas hNIS-transduced tumor tissue could be simultaneously visualized.
Excess iodine decreases NIS mRNA and protein expression [49], reduces radioiodide accumulation into the thyroid and can protect the thyroid for 24 h when administered up to two hours prior to 131I [50]. If the same timing of Ad5/3-hTERT-hNIS and 131I would apply in a clinical setting, extra-iodine administered 4–6 hours after virus injection would saturate the thyroid without hampering transgene-mediated transportation of radioiodide into the tumor after 131I administration (eg. at 24 hours as in our experiments). However, lack of organification of radioiodide into cancer tissues is also a drawback as it reduces accumulation and subsequent dose to the tumor. A rapid radioiodide efflux from tumor cells has been associated with exogenous hNIS-expression [51] and it is clear that this issue is critical in our approach. Another problem may arise from viral oncolysis resulting in the death of the hNIS-expressing cells that limits the duration of the hNIS-expression window. The "timed" expression system used in Ad5/3-hTERT-hNIS, where transgene-expression starts at circa 8 h after infection (and thus closer to oncolysis), may limit the window further. In this regard, a ubiquitous CMV-promoter with high early activity might result in higher hNIS-expression levels [52], [53]. When adenoviral vectors are delivered systemically, they are rapidly taken up by liver Kuppfer cells (KC) [30]. The phenomenon is dose dependent and saturation occurs after high intravenous doses [54] leading to efficient transduction of hepatocytes and other tissues. Adenoviral entry into hepatocytes together with KC activation can result in an immune response that is suggested to be the major determinant of adenovirus induced toxicity [55]. However, if the injected doses are small enough to avoid acute hepatotoxicity, but sufficient to saturate KC, transgene expression in hepatocytes occurs. This provokes a second, adenovirus-protein -specific immunoresponse [56]. Thus, the use of tumor-specific promoters to restrict expression of adenoviral proteins to tumors is important from a safety perspective. Indeed, previous work has shown that hTERT-regulated expression of E1A in an oncolytic adenovirus did not induce liver toxicity [57]. Accordingly, minimal hepatic hNIS-expression was seen in our study.
Human adenovirus is fairly human-specific and thus mice are poor models for studying toxicity. Ultimately, human trials may be needed to evaluate the feasibility and safety of intravenous injection at doses compatible with efficacy. Oncolytic adenoviruses might currently be the best administered intratumorally or intracavitary, perhaps in combination with intravascular delivery. Nevertheless, there are some tantalizing cases suggesting efficacy even after intravenous delivery alone [3], [58]. However, immunogenicity of adenoviruses is an issue with regard to systemic re-administration [59].
In conclusion, Ad5/3-hTERT-hNIS was found to have anti-tumor activity in vitro, and in vivo it extended the survival of mice. Intriguingly, SPECT/CT imaging suggested that hNIS-expression could be detected only in a proportion of the tumor mass, which is likely due to incomplete penetration after systemic administration. Accordingly, combination therapy was not more effective than virus alone. However, an increasing level of hNIS-expression during therapy was detected using SPECT/CT, demonstrating the utility of the method for dynamic imaging of radioiodide therapy in animals with complex orthotopic human cancer xenografts. Before considering the possible clinical translation of the combination therapy to humans, further work is needed for improving tumoral hNIS expression levels and consequently 131I-targeting efficiency [52].
Materials and Methods
Cell lines
Castration resistant prostate cancer cell lines 22Rv1, PC-3 and DU-145 and human embryonic kidney epithelial cell line 293 were purchased from American Type Culture Collection (ATCC, Manassas, VA). PC-3MM2 cells are a metastatic, castration resistant subline of PC-3 (courtesy of Isaiah J. Fidler, M. D. Anderson Cancer Center). 911 cells were obtained from Dr. Alex J. van der Eb (University of Leiden, The Netherlands). All cell lines were cultured as previously described [7]. PC-3 and DU-145 cells have been confirmed to have hTERT-expression [60].
Viral constructs
Wild type adenovirus serotype 5 (Ad5 WT) was purchased from ATCC. Ad5/3luc1 has been described earlier [22].
hTERT-fragment was amplified by PCR from pBT255 (Courtesy of Izumi Horikawa, National Cancer Institute) to create a shuttle vector for E1-region. The following primers were used to result in 361 bp sized amplification product: forward primer, 5′-TTA GCG GCC GCA GAA CAT TTC TCT ATC GA-3′ and reverse primer, 5′-ACC GGA ATG CCA AGC TTA CTT AGA T -3′. NotI-XhoI- digested hTERT-fragment was inserted into NotI-XhoI -digested and calf intestinal alkaline phosphatase (CIAP) -treated pSE1AmpR (constructed previously in the laboratory) to obtain phTERTE1AmpR. Next, both PmeI -linearized phTERTE1AmpR and circular pAdEasy-1.5/3-Δ24 [61] were electroporated into BJ5183 cells for homologous recombination resulting in pAdEasy-hTERT-E1-5/3. DNA from selected recombinant clones was electroporated into DH5α cells for DNA production and further analysis.
To obtain phTERT-E1-5/3-hNIS and phTERT-E1-5/3-Δgp19K, SrfI-linearized pAdEasy-hTERT-E1-5/3 and FspI –linearized pTHSN-hNIS or FspI –linearized pTHSN-Δgp19K [7] were electroporated into BJ5183 cells for homologous recombination. The DNA from selected recombinant clones was electroporated into DH5α cells for DNA-production and further analysis. phTERT-E1-5/3-hNIS and phTERT-E1-5/3-Δgp19K were linearized and transfected into 911 cells using Superfect (Qiagen, Hilden, Germany). The viruses were plaque-purified and propagation was performed on A549 cells followed by standard CsCl purification to obtain a concentration of 4.8×1011 viral particles/ml (vp/ml) and 1.8×1010 plaque forming units/ml (pfu/ml) for Ad5/3-hTERT-hNIS and 5×1011 vp/ml and 5.1×1010 pfu/ml for Ad5/3-hTERT-Δgp19K.
Reverse transcription –PCR (RT-PCR)
Prostate cancer cells were infected with 10 vp/cell of Ad5/3-hTERT-hNIS or Ad5/3-hTERT-Δgp19K for 2 h in 37°C. 24 h and 48 h after infection total RNA was isolated (RNeasy Kit, Qiagen). The samples were treated with DNase before subjected to RT-PCR. 350 ng of RNA was used for each reaction. Amplification was done using OneStep Reverse Transcription –PCR Kit (Qiagen) with 35 cycles and annealing at 54°C. The hNIS-PCR product size (453 bp) was obtained using hNIS-specific primers (forward 5′-CTTCTGAACTCGGTCCTCAC-3′ and reverse 5′-TCCAGAATGTATAGCGGCTC-3′). For ß-actin, (482 bp product size) was obtained using for human ß-actin-specific primers (forward 5′-CGAGGCCCAGAGCAAGACA-3′ and reverse 5′-CACAGCTTCTCCTTAATGTCACG-3′).
Iodine uptake in vitro
1×105 PC-3, PC-3MM2, DU-145 and 22Rv1 cells/well were plated, incubated overnight, and next day infected with 10 vp/cell of Ad5/3-hTERT-hNIS or Ad5/3-hTERT-Δgp19K for two hours. 24 h or 48 h later the cells were washed with PBS and [125I]NaI (MAP Medical Technologies Oy, Finland) was added for 20 min at RT. Cells were washed twice with PBS and 300 µl/well of Cell Culture Lysis Reagent (Promega, Fitchburg, WI) was added. Radioactivity was quantified as described in [7].
Cell killing assays
10,000 cells/well were seeded on 96-well plates, incubated overnight and next day infected with Ad5/3-hTERT-hNIS, Ad5/3-hTERT-Δgp19K, Ad5/3luc1 or Ad5 wild type (0.01–100 vp/cell) for 2 h. The viability of the cells was assessed on day 6 (PC-3MM2), day 7 (PC-3 and DU-145) or day 9 (22Rv1) using the MTS-assay (CellTiter 96 Aqueous One Solution cell proliferation assay, Promega). The cell survival after infection was followed daily with internal controls and the stopped when an almost complete cell killing was seen with the fastest virus [24].
Iodine uptake in vivo
The protocol was identical as described in [7]. NMRI/nude mice (Taconic, Ejby, Denmark) were injected with 7×108 vp of Ad5/3-hTERT-hNIS, Ad5/3-hTERT-Δgp19K or saline.
In vivo survival and biodistribution
NMRI/nude mice (n = 15/group) were purchased from Taconic. 1.5×106 PC-3MM2 cells were inoculated into the left lung of the mice under medetomidine-ketamine anesthesia (day 0) [4], [7]. On day 7, mice were randomized into four groups; mock, 131I alone, Ad5/3-hTERT-hNIS alone or 131I+Ad5/3-hTERT-hNIS and received 5×1010 vp of Ad5/3-hTERT-hNIS or DMEM intravenously. The next day, animals were injected with 36±5 MBq of [131I]NaI (MAP Medical Technologies) in 1×PBS or vehicle intraperitoneally. Three animals from each group were sacrificed 24 hours after the first 131I-treatment for the biodistribution study where radioactivity was measured with an automated gamma-counter (Perkin Elmer Wizard 3, Perkin Elmer, Waltham, MA). Results are expressed as percentage from the initial iodine-dose/g of tissue. 12 mice/group continued in the survival experiment and the same doses were repeated once a week for 3 weeks.
hNIS-expression in vivo by SPECT/CT
NMRI/nude male mice (Harlan, Indianapolis, IN) were treated identical to the survival-experiment. The animals were scanned with SPECT/CT a day after each intravenous Ad5/3-hTERT-hNIS or DMEM injection, three times in total. The 131I (32±6 MBq) or PBS-injections were given after the SPECT/CT-scan.
18±2 MBq of sterile [123I]NaI (MAP Medical Technologies) was injected intravenously under isoflurane-anaesthesia for SPECT-imaging (that was performed 2 h after 123I injections). SPECT/CT was acquired using multi-pinhole nanoSPECT/CT-system with four heads (Bioscan, Washington DC). SPECT-acquisition collected 30 000 counts per projection (16-projections) and the scan-time was 20–25 min. After the third scan mice were sacrificed and the tumors were harvested. All images were reconstructed with MEDISO-software and analyzed using In Vivo Scope-software (both Medical Imaging Systems, Kingsbury, England). Voxel-guided regions of interest (ROI) were drawn using the fused SPECT/CT-images and the results are expressed as percentage of radioactivity in the ROI from the injected activity (at time of the SPECT-scan), divided by the volume of the ROI (in mm3).
Statistical analysis
Student's t-tests were performed to analyze in vitro iodide uptake and cell killing using Microsoft Excel (Microsoft Corporation, WA). Survival analyses were conducted by plotting Kaplan-Meier curves, and the comparisons between groups were done pairwise by the log-rank procedure with PASW statistics 17.0 (SPSS Inc., Chicago, IL). For all analyses, a two-tailed p-value<0.05 was deemed statistically significant.
Supporting Information
Figure S1.
hNIS-expression from Ad5/3-hTERT-hNIS is restricted to tumor cells. A mouse treated with Ad5/3-hTERT-hNIS shows minimal liver 123I− accumulation after the first intravenous injection of the virus (week 1). The next treatments (weeks 2 and 3) did not result in hepatic uptake.
Video S1.
The video shows tumor-specific hNIS-expression on the third treatment week in the lung of a mouse treated with Ad5/3-hTERT-hNIS and 131I−. Some physiological uptake is seen in esophagus, thyroid and salivary glands.
Author Contributions
Conceived and designed the experiments: MR MS TH MT KK AJA. Performed the experiments: MR MS TH MT KK AJA ID VK. Analyzed the data: MR MS TH MT KK AJA. Wrote the paper: MR MS TH MT AK AJA AH.
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- 57. Su CQ, Sham J, Xue HB, Wang XH, Chua D, et al. (2004) Potent antitumoral efficacy of a novel replicative adenovirus CNHK300 targeting telomerase-positive cancer cells. J Cancer Res Clin Oncol 130: 591–603.CQ SuJ. ShamHB XueXH WangD. Chua2004Potent antitumoral efficacy of a novel replicative adenovirus CNHK300 targeting telomerase-positive cancer cells.J Cancer Res Clin Oncol130591603
- 58. Nokisalmi P, Pesonen S, Escutenaire S, Sarkioja M, Raki M, et al. (2010) Oncolytic adenovirus ICOVIR-7 in patients with advanced and refractory solid tumors. Clin Cancer Res 16: 3035–3043.P. NokisalmiS. PesonenS. EscutenaireM. SarkiojaM. Raki2010Oncolytic adenovirus ICOVIR-7 in patients with advanced and refractory solid tumors.Clin Cancer Res1630353043
- 59. Raki M, Sarkioja M, Escutenaire S, Kangasniemi L, Haavisto E, et al. (2011) Switching the fiber knob of oncolytic adenoviruses to avoid neutralizing antibodies in human cancer patients. J Gene Med 13: 253–261.M. RakiM. SarkiojaS. EscutenaireL. KangasniemiE. Haavisto2011Switching the fiber knob of oncolytic adenoviruses to avoid neutralizing antibodies in human cancer patients.J Gene Med13253261
- 60. Huang P, Watanabe M, Kaku H, Kashiwakura Y, Chen J, et al. (2008) Direct and distant antitumor effects of a telomerase-selective oncolytic adenoviral agent, OBP-301, in a mouse prostate cancer model. Cancer Gene Ther 15: 315–322.P. HuangM. WatanabeH. KakuY. KashiwakuraJ. Chen2008Direct and distant antitumor effects of a telomerase-selective oncolytic adenoviral agent, OBP-301, in a mouse prostate cancer model.Cancer Gene Ther15315322
- 61. Kanerva A, Zinn KR, Chaudhuri TR, Lam JT, Suzuki K, et al. (2003) Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus. Mol Ther 8: 449–458.A. KanervaKR ZinnTR ChaudhuriJT LamK. Suzuki2003Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus.Mol Ther8449458


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Trail of Cthulhu is a game written by Kenneth Hite, and published by Pelgrane Press. It uses Robin D. Laws' GUMSHOE system for its underlying engine (i.e., the mechanics the game is built upon), which had previously been used in Pelgrane Press's Fear Itself and The Esoterrorists RPGs. The GUMSHOE system is specifically designed to create stories focusing on investigative mysteries and thus is perfectly suited for exploring the setting based upon the writings of H.P. Lovecraft and his emulators. ToC retails for $39.95 for the hard cover version and $19.95 for the PDF version. I am reviewing the hard cover book.
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Just in case you're not familiar with the Cthulhu Mythos…
The Cthulhu Mythos milieu focuses on mankind's interactions – whether they be ordinary citizens, dark sorcerers, or insane cultists – with primordial alien races, dark gods, and other ancient beings that we were not meant to know. As such it's generally a very dark and grim setting, where insanity, death, or worse await those who delve too far into the details of the Mythos. The basic idea both in H.P.L.'s writings and in the game itself is that ignorance is bliss and knowing too much can shatter a person's mind. As such, the setting is one where PCs' lives can be very short indeed, especially if one sticks to the tone established in the majority of Lovecraft's stories (Robert Howard's stories tend to have more of a pulp-tone, in which investigators fight the horrors using weapons).
A bit of nomenclature: keeping true to its Call of Cthulhu roots, player characters (PCs) are known as Investigators in the game and the Gamemaster (GM) is known as The Keeper. I'll be using these terms extensively in the review below.
Physical Details
The book is beautiful, with a tight binding and an attractive, very evocative, color cover. It's printed on high quality paper with a gray-scale interior, although page headers, dividers, frames, and markers are done in a brassy-brown tone which adds a nice antique effect that fits the material well.
The layout is done in a narrow, three-column form which looks attractive but tends to make the pages feel really dense. It also creates some rather cramped lines at times, something that's exacerbated by a few editing/layout gaffs that lead to spots where words have no real space between them (this is particularly problematic with the italics) or where bullet points aren't indented, causing them to blend into the text above and below the list. This is evident particularly in the tables and sidebars. Similarly, while the book's editing is good, it could have used another couple passes of a careful proofreader since there are missing words and other typos still evident. All of these criticisms are minor points though since they are hardly common nor problematic, and taken as a whole, the book is very well edited and laid out.
The artwork, all done by Jérôme Huguenin, is for the most part excellent, and in places downright amazing. I realize that others don't share my opinion, as a quick search of the internet will turn up all kinds of opinions – the chief complaint is that some of the artwork looks murky or smudged, and indeed it does. However, I think this style actually captures the essence of Lovecraft's own approach: he often described his creations in very vague terms and the art in the book captures the spirit of those descriptions by presenting the basic form of the creatures and leaving the details to the imagination. It's hard to dispute that some of the art is simply fantastic.
One bit about the layout deserves special mention: throughout the book, a pair of symbols are used to denote rules or material that are particularly well-suited for either a "Purist" or "Pulp" play style. As I mentioned previously, Cthulhu Mythos stories come in two basic flavors. In one, the investigators are generally up against creatures and forces beyond human comprehension or influence (i.e., you generally flee for your life and a good outcome is one where you make it home alive and still sane) – that's "Purist" in ToC nomenclature. In the other, characters struggle against the forces of the Mythos and in some cases can fight back (i.e., you pull out your Tommy gun and dynamite and shoot Deep Ones) – that's "Pulp." ToC does not favor one style of play over the other, and instead provides rules that work for both, supplemented by material, optional rules tweaks, and GM advice denoted in the text by the two symbols. I love this approach since it both widens the variety of play the game supports and allows the group to tailor the game to their particular tastes and needs.
The Book Contents
The book is well-written, with a clear, conversational tone which is both scholarly and easy to follow. Kenneth Hite is well-known for his knowledge of H.P. Lovecraft's work, and that comes through loud and clear in the tone of the book which offers analysis of both Lovecraft's works as well as how they can be translated in to the game. For those coming from a Call of Cthulhu (CoC) background (whose core products were set primarily in the 1920s), ToC moves the timeline forward about a decade and uses the 1930s as its default time period. While this choice may seem odd or even arbitrary at first glance, Hite explains in his typical logical and scholarly manner why he chose the 1930s, and his rich and frank analysis of the time period and really helps sell the reader on the decade as a great place to set a ToC campaign.
After the requisite introduction – in which the ubiquitous "What is roleplaying?" section is thankfully not present – the book moves directly into character creation which leads players through the process of building an investigator.
Characters are built based on a point-buy system in which characters are defined first by an occupation (including archaeologist, artist, clergy, dilettante, and even hobo). The choice of occupation then helps determine the types of Abilities (more on these later) the character can buy at a discounted rate, as well as the limit of their credit rating (naturally a dilettante has access to more money than a hobo) as well as any special abilities unique to that occupation. Characters are also defined by a personal Drive, which is what motivates them to be an investigator, as well as one or more Pillars of Sanity, which are the principles that the investigator relies on to maintain their mental health.
After character creation, the book dives into the actual rules of play in the Clues, Tests, & Contests chapter. What makes ToC – and GUMSHOE in general – great for busy adults is that it is a relatively rules-light system since it uses a unified system to determine success and failure at various tasks. This means it is easy to learn and therefore it takes little time to go from reading the book to playing.
The remainder of the book is dedicated to background information about the Mythos, GM advice, a sample adventure, and various appendices.
The Rules
It's all about the investigation
Trail of Cthulhu is built upon the GUMSHOE system (created by Robin D. Laws), which was first used in Fear Itself and since then has been used for multiple games, including The Esoterrorists, Mutant City Blues, and the newly released Ashen Stars. GUMSHOE is specifically designed to be used in investigative games, where the characters are finding and interpreting clues in an attempt to solve a crime or problem. As such, it's a perfect fit for the typical Cthulhu scenario which involves investigating supernatural phenomena, locations or crimes.
The basic premise of GUMSHOE is that investigative skills (i.e., those you use to find or interpret clues) do not rely on a random chance for success. Instead, a character with an appropriate skill is guaranteed to find the core clues necessary for them to move forward in their investigation. Gone are the days where a single failed skill check would lead to a dead end for the party. Each character has a number of investigative abilities, representing their specialties and areas of knowledge. The goal is for the group as a whole to be able to cover all the important bases collectively.
However, there's more depth to the game than simply a "if you have the skill, you succeed" because each character has points assigned to these skills which can be spent to gain additional information in a scene. These secondary clues are meant to add more details to the core clues, providing insight in to the overall plot, revealing information pertaining to a side story, or perhaps speeding the investigation by allowing the players to bypass some obstacle, location, or requirement. These points only refresh at the end of the adventure so players need to manage their use carefully. Thus, what you have is a very simple "automatic success" mechanic in terms of finding clues, and a resource management mechanic for determining success at finding additional information and making a character shine in a particular scene.
Besides investigative abilities, characters also have General Abilities which are used for your typical "skill test" situations. Characters have a number of these skills, which range from Athletics to Piloting to Stealth. General Abilities have ratings which provide a pool of points to spend from, but these expenditures work somewhat differently because they are used to modify a roll. For these tests, the Keeper sets a difficulty for a given task (the default value is 4, which is used in most situations), and a player then decides how many points to spend from the appropriate Ability's pool. This is then added to the roll of a single six-sided die, and if the result is equal to or greater than the set target the character succeeds. This is a simple and elegant mechanic which means that the average, untrained character succeeds at most things 50% of the time and that training in a relevant ability then modifies this. Once again, it's part resource management, part traditional chance which fits the genre and setting very well. One of my favorite touches is the fact that the most useful General Ability in the game is Fleeing.
Contests and combat work in a similar way, using opposed tests. Damage is handled using a traditional "hit point" type system, and keeping true to the original CoC RPG, ToC also includes a mental health system via the Stability (short-term stress level) and Sanity (long-term mental health) stats which I will discuss in more detail later. Combat itself is very simple and streamlined, opting to emphasize narration and story rather than "tactical" decisions but there is enough crunch to keep things interesting. The one criticism I have about combat is that the firearm rules contain a bunch of weapon-specific rules (e.g., there are a few different rules specific to shotguns) which can be a bit overwhelming at first, especially since they're spread out over a number of pages. However, compared to the average RPG, these types of exceptions are quite minimal and any group should have them mastered with just a few games under their belts.
Rules for magic use are also included, although these are a bit difficult to find at first, because they're located in the Cthulhu Mythos section, sandwiched between the "Gods & Titans" and "Creatures" sections. This placement is somewhat awkward from the PC perspective, but ultimately makes sense for the "traditionalist" in which spells, tomes, and magic in general are supposed to be dangerous and a bit of a mystery and therefore are something that the Keeper is aware of but the players are not. The rules also provide ways of handling disease, illness, recovery, and advancement. All of these are simple, straight-forward rules that are built off of the core mechanics.
One design choice of GUMSHOE, and thus ToC, is that most rolls are "player facing" meaning that NPCs never roll independently of the PCs to determine success or failure – instead players pick up and roll the dice to determine the outcome of most situations, either testing one of their skills or making an opposed test versus the NPC. For example, an NPC doesn't use Stealth – the PC uses his Sense Trouble ability. While this may seem like an odd bit of minutiae, it has the effect of putting success or failure into the hands of the players and means their fate is largely determined by their rolls, rather than a series of mysterious rolls hidden behind a screen. It also keeps players engaged and streamlines a lot of ordinary situations by eliminating a bunch of extra die rolls and stat referencing in determining success or failure: it doesn't really matter what the shoggoth's Stealth ability is, because you're only using the PC's Sense Trouble ability. If this seems like a recipe for disaster where PCs need to "have a point in everything" to be viable, keep in mind that the ranks in Abilities are only used to optionally modify rolls, and so even an "untrained" PC gets to test any particular ability. Thus, investigators are pretty competent at just about everything and at least have a shot of success in most situations.
Going Insane – The Descent into Madness
Like its predecessor, ToC uses a Sanity score, and many events in the typical session will threaten the investigator's fragile grip on reality and thus their Sanity score, driving them toward eventual post-traumatic stress or full blown insanity. However, unlike most other games, ToC actually breaks mental health into two separate stats: Stability and Sanity. Stability represents a PC's resistance to mental stress and their ability to handle short-term, acute stress. Sanity, in contrast, is a measure of a PC's ability to handle the mind-blowing revelations associated with the Cthulhu Mythos and remain in touch with ordinary human concerns – it's all about the long-term spiral into madness associated with knowing "too much" about what's really going on. While Stability returns after each adventure, Sanity, once lost, is never recovered.
Stability and Sanity also interact with other PC characteristics, specifically a character's Drive (what motivates them to investigate the Mythos) and Pillars of Sanity (what ordinary things anchor them to their human existence). Drives function in a carrot or stick fashion, pushing the PC forward in certain situations and providing a bit of reinforcement for behaving in ways consistent with that drive or punishment when one goes against one's primary motivator. Pillars of Sanity, of which each character has one or more, serve a similar type function, providing the character something story-wise to rely upon to maintain their own sanity as well as providing the Keeper with some character-relevant details to incorporate into stories or even threaten.
In my opinion, this dual mental health model is particularly ingenious since it allows for a PC's mental health to slowly erode over a series of adventures via the Sanity stat, while allowing acute stressors within a particular investigation to be handled via the Stability stat. This does not mean your character can't go insane during a single adventure – they most certainly can – only that running up against the Mythos in the form of creatures, tomes, or Great Old Ones results in accumulated stress that is tracked separately from your day-to-day, or more accurately adventure-to-adventure, stress levels. As such, ToC is geared towards a slower descent in to madness than Call of Cthulhu, which makes it better suited to long-term campaign play.
I also think the system models the "shock" versus "horrific realization" mixture of events that a typical Cthulhu investigation involves much better by separating "ordinary" bad stuff from cosmically mind-blowing revelations – these differing elements are part of H.P.L.'s writing as well (e.g., contrast the Shadow Over Innsmouth's narrator's fear during his escape from the town versus his realization of his own patronage afterward) and thus I think the game captures this dichotomy very well. In addition, each character's Pillars of Sanity and their personal Drive interact with these choices, making for a very rich system that really makes investigators feel "real" to me.
Of course, there's a bit of a downside to all of this: the Stability/Sanity rules are easily the most complicated system in the game and take some effort to really learn because the two interact with each other – Stability losses caused by running up against Mythos encounters can also lead to Sanity losses and how these interact is a tad bit fiddly. However, like the weapon rules, these issues are quite minor when compared to other RPG systems. In addition, the ToC Keeper's Screen is a valuable resource in these cases since it summarizes the Stability and Sanity loss rules on one of its panels, and thus is highly recommended.
Explaining the Unexplainable
More than half the book is dedicated to explanations of the Mythos including its Gods, cults, monsters, and magic. This includes an awesome breakdown of the Cthulhu Great Old Ones in which Hite provides a variety of alternate explanations for each of the major "Gods." These are based on the various Mythos stories, as well as some more modern twists, and provide the ability for the Keeper to tailor the Mythos to his or her own tastes and needs. All of the major Mythos creatures are also described in some detail, including game stats since these are the types of monsters investigators can actually fight.
Details on the spells and major tomes are equally excellent. The rules for using tomes and magic are fairly straight-forward and accurately represent what occurs in the literature that inspires the game and genre: magic is something that one delves into very cautiously and ultimately there is a price to be paid for its use. In particular I love the explanations of the various tomes since they're both interesting and inspirational.
The Mythos section of the book wraps up with descriptions of the major cults associated with the Outer Gods, providing a description of each group, their locations where they're likely to be active, and a set of hooks that a Keeper can use to incorporate the cult into their campaign. These are all well-written (and researched since most of them come straight out of the source material) and are there's a lot of information that can be mined from this section as inspiration.
The '30s – Setting & Background
As mentioned above, ToC uses the 1930s as its default setting, and the author provides a really detailed overview of the the events and culture of the 1930s to provide the reader with some insight and inspiration about the time period. Of course, the section is fairly brief and any good Keeper is going to want to consult some outside material to provide further details, but even this is made easy since the book provides some recommended sources to start with. In terms of what is provided, actual historical events are interwoven with fictional material culled from the various Mythos stories seamlessly and it's a very interesting read. This includes an overview of some of the major "hotspots" around the globe, providing both historical material and Cthulhu Mythos teasers and hooks for the Keeper to work with. One of the advantages of advancing the timeline in ToC by roughly a decade compared to the H.P.L. stories is that all of those events happened in the past, providing some historical Mythos events to build the campaign upon.
Wrapping Things Up
The remainder of the book consists of player and GM advice along with a set of sample "Campaign Frames" which are essentially thematic premises or concepts upon which a series of adventures can be built. Three are included in the book, with each providing an overview of the setting, style, Mythos details, key NPCs, and possible rules variations that can be incorporated in to the campaign. I have to say these are cool and evidently others, including Pelgrane Press, agree since two out of the three presented in the core rule book have since come out as fully fleshed-out supplements (The Armitage Files and Bookhounds of London). What I love most about the three that are presented is that each is completely different: one involves a purist-style group from Miskatonic University, another Pulp-style X-Files sort of government team, and the third a group of unsavory rare book dealers based out of London.
The book wraps up with a sample adventure, "The Kingsbury Horror," which is set in Cleveland and was inspired by a real historical event.
The Verdict
By now it should be evident that I am a huge fan of Trail of Cthulhu. I think it manages to capture the feel Lovecraft's stories, particularly when played in Purist mode, with rules built to complement the stories. GUMSHOE is a perfect fit for investigating the supernatural mysteries and horrors associated with the Cthulhu Mythos. It also is simple enough to be run in a more "off-the-cuff" improvisational style and doesn't require a great deal of prep on the part of the GM, an important consideration for those of use with busy lives.
Another factor that makes ToC simply a great RPG is the quality of the supplemental material that's been published to support the system: all of the adventures published to date are excellent as are the other supplements. While none of the material is essential, all of it is very interesting and useful, with a clear aim of providing support rather than providing an endless series of splat books. I would also be remiss if I didn't mention that the huge volume of material that Chaosium has published over the past three decades can also be used with ToC with some minor conversion work, for which ToC provides guidelines.
In the end, Trail of Cthulhu is a roleplaying game I would highly recommend to anyone who is interested in Lovecraft's stories, or horror investigation in general. The game's system, setting, and supplemental material all work together to create an interesting and thoroughly compelling world to explore.Go Back to Top. Skip To: Start of Article.

subset: commoncrawl


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An Exclusive Interview from the Chairwoman of the International Service Design Network, Birgit Mager
Since 1995 Birgit Mager has held the first European professorship in service design at the University of Applied Sciences in Cologne, Germany, and since then has developed the field of service design constantly in theory, methodology and practice. Her numerous lectures, publications and projects have strongly supported the implementation of a new understanding of the economical, ecological and social function of the design in the domain of services. The following is an interview conducted in the context of the World Industrial Design Conference in China.


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Triple Digit Temps Hit AZ
PHOENIX (AP) _ Forecasters warn that much of the northern half of Arizona will be ripe for wildfires Tuesday amid strong winds and low humidity.
The National Weather Service has posted red flag warnings starting at 11 a.m. for the region, with humidity expected to drop into single digits while wind gusts are set to top out at 40 mph.
The wildfire risks come after the mercury hit triple digits in Phoenix for the first time this year. Temperatures in the city topped out at 100 on Sunday.
National Weather Service forecaster Keith Kincaid says the last time Phoenix had a 100-degree day was last Oct. 4.
Meteorologists say the heat is expected to offer up a couple more triple-digit days before the weather cools off to the mid-90s by mid-week.


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BIG Punch
NOTICE: Heil HC-4 and HC-5 elements are no longer available from Heil Sound. W4RT has NO, repeat NO, HC-4 or HC-5 elements left in stock.
One BIG Punch (OBP) is a custom add-on accessory for the Yaesu MH-31 microphone that is used with the FT-817(ND), FT-847, FT-857(D), and FT-897(D). The OBP is an AF-based speech compressor specifically configured to provide remarkable increase in talk power while maintaining good audio quality (articulation) for communication purposes. Measurements using a stock FT-817 and a MH-31 containing the One BIG Punch showed that the average talk power increased by about 5 dB over the unmodified MH-31. When the optional SSB filter (Collins Mechanical Filter) for the FT-817 was used in conjunction, the talk power increased another dB or so. This information is provided for general expectations, not as a firm specification.
Want Even More PUNCH?
Then have W4RT Electronics install both the HC-4 (OBP active) & HC-5 (OBP not active) Microphone Elements in your MH-31 with the One BIG Punch! See ordering selections below. Some users like using the HC-5 on FM and the HC-4 with the OBP on HF. Overall field usage by many users determined that the HC-4 with the OBP (and with the optional SSB filter) was the best combination on HF to successfully make QSOs under marginal situations (QRP typical). NOTE: Heil is no longer offering the individual elements. W4RT Electronics has a VERY limited number of these elements left in stock.
W4RT Electronics also offers an OBP Module tha has the One BIG Punch, Heil HC-4, and Heil HC-5 microphone elements fully integrated together and ready for you to install. For those of you that just can be off the air for a few days while W4RT upgrades your MH-31, you can simply order the fully assembled MH-31A8J with the OBP Module. It just doesn't get much better than this!


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Hey Everyone,
Im on the final stretch before I release my first iPhone Application. It's an iPhone Application to monitor your Envato Marketplace sales.
Im looking for about 5 people to test the application using their own Envato account, via API key access.
Criteria are:
- You have an iPhone or iPod Touch and have access to iTunes
- Have at least 5 or more sales a day on average (the more the better!)
- You more than a couple of products
- Can provide useful feedback about usability, design and functionality of the application
So how do you register? Easy just drop me an email.
- Your Envato username
- Approximate amount of sales per day
- What device you'll be using (iPhone, iPhone 3G, iPhone 3GS or iPod Touch)
I will stop accepting registrations on the 23rd February
Once I've got a few people on the app and it is stable it will be ready to release! The faster you get in the better chance you have, and the sooner the app is released
Thanks Everyone,
Alex Eckermann
Hey blabus, I'm interested in your application. Please send me your specs and we'll go from there.
Im interested too. Maybe we could both work on this?
Im just curious, does anyone here make templates or items for the Envato "mothership" full time?
Ive seen a few users with badges indicating they are making a reasonable income. I do mine for fun really, upload them and see what happens, but I would imagine its a bit dicey doing it full time.
One of mine had the same problem. It too is showing the high res image (its been resized as well.) instead of my custom 590px wide.
Yes. In ThemeForest, a sister site, you can change anything from description to the downloadable files if you've messed something up, so I would bet (without looking) that you can do that here too. No need for re-moderation for simple things like description but I think changing files needs a check before it can go up.
Edit: beat me to it ranfirefly!
Just in the past 10 or so minutes ive had some strange graphics problems, probably stylesheet related. After a refresh they went away. I took screenshots just in case they help you out.
http://img.skitch.com/20090209-mihr3fms5amufti6kdgscqk3an.jpg http://img.skitch.com/20090209-tp1px7p8ti26h6ce5q2in42m4c.jpg http://img.skitch.com/20090209-mbu7diypetn35bf63qsa4u1kcc.jpg
Awesome! Do you know what item put it over the mark?
Ive made a few of those files But like xkey said the logos and trademarks are not used.
I've just googled for 'stock icons' and the first one I went to was selling icons that looked identically like some new model cars but they did not have any trademarks on them.
We'll see what happens when the Site Manager or Moderator comes across this post.
Hi, Here is a similar post from another user: http://themeforest.net/forums/thread/sifr-font-licensing/8849
I would say that you wouldn't be able to use the font, embedded or not, unless you have the right/license to. Free fonts are the way to go but always check the licensing for them.


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Help - My hair is lifeless, dull, faded
I've been coloring with a semi-permanent and it's such a hassle (and might be causing hair loss), so I want to stop. But I have mousy brown hair and I'll look washed out. Women with dark hair who go silver look gorgeous. Are there any brown-haired gals out there who've gone gray? How do you do it gracefully? And what do you use to counteract dryness and give your hair more shine?
I've used JessiCurl gentle cleansing cream, Aubrey's Organics green tea shampoo for clairfying, DevaCurl Low Poo, and some other non-sulfate cleaners. For conditioning, I've switched around with Aubrey's Honeysuckle and Rose, Trader Joe's Tea Tree Tingle, DevaCurl One Condition, and Recoil.
Another mousy brunette going gray here (though mine started in my early 20's, thanks mom!).
I think how gracefully it happens it all depends on how your grays are coming in. For example, I would have loved to rock a single, chunky streak of gray hair in my 20's. I would have even accepted it in highlight kind of streaks. Instead I got classic "salt and pepper" grays - just dozens of random gray hairs all over the place. So naturally I started dying it.
Back in January, I switched from box dyes to henna and my hair health has improved dramatically since then! It's shiny, more curl pattern has gotten stronger and I think it may even be coming in a bit thicker. The first couple of applications felt like they were drying my hair out, but since I switched to a CG conditioner that hasn't been an issue.
I was thinking about henna, but not sure I could do it myself. So it doesn't dry out the hair? What conditioner do you use? Thanks!
I am permanently dyed so hair is porous/ damaged, get shine from hydrolysed protein, flaxseed gel, natural oils. For dryness I use piles of leave in conditioner, panthenol and ceramides. Choose your ingredients and products based on your hair properties and dew points/ humidity.
It feels like that for the first few days for a lot of people especially those who let water drench their hair fewer than once a week.
If you are grey you need to be careful how you use it. If you don't leave it on long enough (minimum 4 hours) you can get orange hair.
There is some good general henna info on this blog here and here
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Any Post Ban gun in NY does not meet the NY AW defination. So my ar is not an assault weapon. We have to wait to see the newest definition of AW in NY, bet its a doozie.
Thing that really troubles me is that NYC already has a more strict version of the AWB than NYS.
NYS has any 2 features. NYC has any 1 feature thereby making AR's and AK's immediately illegal even if they are legal everywhere else in the state.
Also long arms in NYC can only have a 5 round mag capacity.
So theoretically if this is true what are they planning on confiscating? There already should be no legally owned firearms that could potentially be made illegal under a new law for them to try and take. Unless they are planning on coming after any semi auto rifle or handgun which is very troubling indeed...
Cops killed while confiscating guns - thats exactly what feinstein&co are dreaming for.
If such thing to happen, the public outcry will let the feinsteins to ban ALL guns except maybe hunting single-shots and airrifles.
And then the country is doomed.
I dont believe confiscation is going to happen. With the fiscal shambles this state is in, we would be irresponsible to try and spend millions on taking guns away.
The agenda is to disarm the citizenry and make everyone totally dependent on the government. Bow down to your president or should it be dictator ?
Write your congressmen people.....beat down their door...The only way is to let them know that legal gun owners are the ones that vote...criminals and wackos do not...if they get their agenda passed we are all going to take a big bite of a **** sandwich, and the bad guys will be drooling....great state and country we live in....the 99 % good have to be punished for the 1 % scum
NYC's AWB is stronger than NYS's. I think as strong as the possible new AWB we might get nation-wide.
For example: ARs and AKs are legal in NYS, but not NYC.
Its actually amazing that we can continue to be headed toward every right that an American has to be taken away , something as dumb as taking larger soda off the shelf was just another control issue that has gotten approved . The Final thought on this they in the end just want the American citizen to be completely needing on the government for everything or they won't be happy .. I get sick to my stomach every time I have to hear the President or some sort of congress talk ... I has become a terrible joke that will only get worse over time .
My predictIon since the state is broke, they'll package it as a budgetary item.. You get grandfathered in with your "assault weapons" but pay a yearly fee for "record keeping".
I love the south
My perdiction is that I will move out of this rotten state...


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- Join Date
- Feb 2004
- Location
- Toronto
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22nd Annual Toronto Supra Club Meet Aug 18-20
Please mark your calendars.
We are anticipating another great year for our meet.
We will be posting all information on our Facebook page. Please check there for details.
This is our annual meet tshirt. Please order early as we will have a very limited supply at the meet.
Tshirts are $20 each. please indicate quantity and size. payment can be made through email@example.com
Look forward to seeing everyone there.
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