The objective of this experimental study was to develop a new combination technique for electrode placement and the histomorphological evaluation of its effectiveness for the electrochemical lysis of large intraocular tumours.The ECL was conducted on two freshly enucleated eyes containing large tumours, with maximal prominence of 11 and 12 mm and maximal base diameter of 16 and 19 mm, respectively. The ECL was carried out using an ECU-300  apparatus generating an electrochemical charge of 30-35 K. In the course of the ECL procedure we used a new original combination technique of electrode placement, i.e., the anode was a surface electrode and the cathode was an intrastromal electrode. The anode had an original design.A greyscale B-scan performed after the ECL completion showed decreased echogenicity and heterogeneity of the echo-structure of the tumour. According to the bioimpedancemetry data, the average duration of the ECL session was from 20 to 30 minutes depending on the tumour size. The results of pathomorphological examination performed after the ECL on two freshly enucleated eyes appeared to be similar. Thus, in both cases after lysis the eyeball did not change its size or shape. In both cases the tumour originated from the choroid plexus and showed subtotal necrosis. There was a pronounced boundary between the intact and electrochemically damaged tumour regions which attests to the local effect of the ECL restricted to the electrode placement area only.The growing interest in the ECL procedure is due not only to its availability and low cost but mainly to its real clinical effect demonstrated in numerous publications. The absence of a developed ECL technology for the treatment of intraocular tumors, and, hence, reports on its clinical effectiveness, gave us the impetus to conduct this study.The proposed ECL method is promising and can be considered as optional for the organ-sparing treatment of large-sized intraocular tumours. Further optimization of the ECL parameters, as well as the development of sets of surface and intrastromal electrodes for different types of tumours, is required. Choroidal melanoma (CM) is the most common primary malignant intraocular tumour that accounts for nearly 80% of all choroid plexus tumours. Owing to the high risk of metastases 3–16%) [6% 1–4],,4], the Nowadays, the preferred concept in ocular oncology for CM treatment is the use of organ-sparing methods which basically require radical surgery against the tumour while causing minimal damage to surrounding normal tissues.Currently, the range of organ-sparing methods available for CM treatment is wide enough and includes laser photocoagulation, brachytherapy, cryodestruction, transpupillary thermotherapy, photodynamic therapy, surgical resection of the tumour (en block resection), etc. .The possibility of the use of organ-sparing treatment of CM will largely depend on the tumour size  and location . Enucleation is performed to remove large-sized eye tumours.In view of the above, the development of new minimally invasive and organ-sparing methods for the treatment of large CMs  is becoming a topical issue.A good example of the organ-sparing trend in oncology is the electrochemical lysis (ECL) method which induces destructive chemical reactions occurring during direct current flow between two bipolar electrodes introduced into the tumour (HCl is produced at the anode and NaOH at the cathode), that subsequently results in tissue coagulation and colliquative necrosis around the electrodes.The ECL method has been fairly successfully used for the treatment of breast cancer, hepatic carcinoma, and tumour metastases in the liver, benign prostatic hyperplasia, cancer of oesophagus, lungs, pancreas, and skin –11.In general oncology, the standard ECL technique involves a parallel introduction of two or more needle electrodes into the tumour. Using a similar approach in ocular oncology, the electrodes should be introduced in the intraocular tumour ***transsclerally in the zone of projection of the tumour base onto the sclera. In order to obtain an adequate necrosis of large tumours, three or more electrodes must be introduced intrastromally and correctly positioned under ultrasound control  which is fraught with risks and several complications, such as iatrogenic retina tear, hemophthalmos, subretinal and subchoroidal haemorrhages, etc.Therefore, the problems with electrode placement and inability to predict the optimal electric field’s effect on the tumour make the search for new approaches to ECL application in ocular oncology a topical undertaking.The objective of this experimental study was to develop a new combination technique for electrode placement and the histomorphological evaluation of its effectiveness for the ECL of the large-sized intraocular tumours.The ECL was conducted on two freshly enucleated eyes containing the large-sized tumours, with maximal prominence of 11 and 12 mm and maximal base diameter of 16 and 19 mm, respectively.The ECL was carried out using the “ECU-300”  apparatus generating an electrochemical charge of 30–35 K. In the course of the ECL procedure, we used a new original combination technique of electrode placement, i.e., the anode was a surface electrode and the cathode was an intrastromal electrode. The anode had an original design: it was made of the platinum wire to form a round mesh of 9 mm in diameter and with a hole in the centre. The cathode was a needle electrode made of 0.5 mm platinum wire .At the ECL preparation step, we determined the boundaries of the tumour base projection onto the sclera by their marking with 1% water– alcohol solution of brilliant green. To perform the ECL, the anode was applied onto the sclera within previously marked boundaries of the tumour base and stitched with two interrupted sutures. The cathode was introduced perpendicularly to the sclera into the centre of the tumour base through the hole in the anode electrode. To introduce the cathode into the tumour, a trocar with a screw-type length adjustment and a 25-G cannula were used.The length of trocar was adjusted to accommodate the length of the extra-scleral part of the 25-G cannula + sclera thickness + electrode penetration depth into the tumour. Then, sclerotomy was performed using the trocar inserted into the lumen of the cannula, by inserting it to its full depth into the tumour perpendicularly to the sclera followed by trocar withdrawal from the lumen of the cannula and its replacement with the electrode of a predetermined length.The depth to which the electrode was inserted into the tumour had been determined in advance based on the results of previous ultrasonic scanning  by subtracting 1.5–2 mm from the value of the maximum prominence in the centre of the tumour. The length of the electrode active part was calculated in the same manner as the trocar length .The active positioning of the intrastromal electrode was carried out in the course of surgery under the trans-corneal and trans-scleral ultrasound control using a 10-MHz probe of the Ultrascan  apparatus.Z) was conducted in an automated mode, and the software produced the chart of changes of tissue resistance in real-time (Z) flattened out and appeared to be hardly affected by time, it was an indication for completion of the ECL procedure.To evaluate the ECL effectiveness, we used the bioimpedancemetry method which is the measurement of total electrical resistance of tumour tissue placed between two electrodes in the direct electric field of variable frequency. Multiple impedance measurements of lysed tissue in the course of ECL were performed using an experimental setup at 2 and 10 kHz. To this end, the ECL procedure was interrupted every 3 min for 1–2 s. The same electrodes were used for both ECL and bioimpedancemetry. Impedance measurement . In the centre of the area there was a canal resulted from the insertion of the intrastromal electrode (cathode), its opening containing dark fluid expelled after the electrode withdrawal. When the eyeball was cut open, the impurity-free liquid of the vitreous body leaked out through the base of the tumour. The location of eye integuments and tumour was in accord with the results of clinical and instrumental examination. On section, the tumour is dark and has small slit-shaped spaces through which a slightly frothy, gel-like liquid, with admixture of reddish-brown, blood-tinged fluid, is oozing.In both cases the tumour was originated from the choroid plexus and showed subtotal necrosis.For the sake of convenience of description of the ECL-induced tumour morphological changes, the tumour was conditionally divided into three parts: the apex, middle part and periscleral part.The apex part showed necrosis with cell fragmentation, nucleus contractions and stiffing (caryopicnosis) and nucleus fragmentations (caryorexis), pigment condensation, appearance of slit-shaped spaces in place of the vessels filled with lysed blood and gaps along the contour of the palisade structures .The middle part demonstrated total cell necrosis. The contours of the gaps resemble those of individual cells and lumens of destroyed vessels . The sepIn the periscleral part of the tumour located next to the cathode canal the morphological picture resembled that of the middle part except for the smaller size cavities, owing to the absence of large vessels in this part of the tumour .The lumen of the canal formed after withdrawal of the intra-scleral cathode electrode) is filled with pigmented debris with an admixture of lysed blood .Given that the area of the surface electrode is smaller than the tumour base area, some periscleral spots of the intact tumour tissue have been found. These spots were characteristic for CM, i.e., they were intensively pigmented and composed mainly of spindle cell type A melanocytes which showed moderate polymorphism and minimal infiltration of the inner layers of the sclera.There was a pronounced boundary between the intact and electrochemically damaged tumour regions  which atThe growing interest in the ECL procedure is due not only to its availability and low cost but mainly to its real clinical effect demonstrated in numerous publications , 7–14.The absence of developed ECL technology for treatment of intraocular tumours, and, hence, reports on its clinical effectiveness, gave us the impetus to conduct this study.We set out to develop a new method making use of combination of the surface and intrastromal electrodes, as well as their original placement, which hitherto has never been described. To evaluate the ECL effectiveness, we performed the bioimpedancemetry of the ECL-treated tissues and morphological examinations of the large-sized intraocular tumours. The experiment was aimed at developing an integrated and manageable ECL method based on unbiased impedancemetry measurements, thus allowing the evaluation of its dynamics and determination of the point of its completion.The direct current flow between the electrodes results in tissue devitalization owing to its electrolysis. In the course of the ECL procedure the resistance of tissue (Z) positioned between the electrodes drops indicating the occurrence of tissue necrosis. The registration of therapeutic effect  is based on the impedancemetry measurements which are consistent and little changeable with time.The histological picture of the ECL-induced CM necrosis demonstrates different patterns of tumour destruction, primarily of its vessels, around each electrode depending on their polarity.Under the cathode electrode a pronounced vasodilatation and engorgement of large vessels occur accompanied by the capillary wall destruction and extensive hemorrhages into the necrotized tissue due to an increased turgor pressure caused by the electro-osmotic fluid flow. On the anode side the capillary reaction was only slightly noticeable. Therefore, the electromagnetic field effects in biological tissues are due to the obstruction of the microvascular bed. In the cathode region the capillaries are blocked owing to the electro-osmotic fluid flow, and in the anode region the pathological changes occur due to the micro-thrombotic events. Owing to the remote placement of the electromagnetic field source the final area of the ECL-induced tissue lesions must exceed the total area of primary necrosis.The presence of the periscleral sites of intact tumour separated by a pronounced boundary from the electrochemically damaged tumour spots indicates to the importance of the accurate placement of surface electrode, as well as its selection, taking into account the size of tumour base projection onto the sclera and avoiding the use of electrodes with smaller contact area.One of the particular features of ECL treatment of the intraocular tumours is the increased intraocular pressure occurred in the course of the ECL procedure. This is due to active gas bubble formation within the tumour and hampered evacuation of liquid debris via the electrode canal. Therefore, the removal of cell-free products of tumour necrosis using the vitreotom secures the maintenance of the original level of intraocular pressure, does not interfere with the ECL procedure and enables process stability owing to the replenishment of evacuated volume with BSS saline.The updated pattern of the electrode placement geometry using surface extrascleral and intrastromal electrodes and adherence to correct polarity while placing electrode in the tumour, opens up new horizons in achieving total intraocular tumour destruction. The design feature of this ECL technique is an individual selection of surface electrode which covers the whole tumour base over its projection onto the sclera, while a centrally positioned hole enables to introduce the intrastromal electrode to any depth allowing its placement maximally close to the tumour apex and preventing electrode dislocation within this position.When setting the parameters of ECL, we were guided by the charge value which would knowingly cause necrosis of a given tumour volume. This value was experimentally established to be 30 K per 1 cm3 of tumour tissue, and the increase of charge above this value does not practically cause the expansion of necrosis zone , 16. HowFurther modifications, refinement and customizing of the ECL method to address the specific needs of ophthalmologic oncology will allow the pre-modelling of tumour necrosis patterns aided by using the specifically designed software, and thus will help to achieve higher treatment effectiveness. Working out of the objective method of real-time assessment of pathologic changes occurring within the tumour, e.g., by measuring active and reactive tissue resistance by means of bio impedancemetry, provides the effective control and regulation of the ECL procedure.Also, the above method offers additional benefits in investigating tumour morphology and growth pattern, as the fine-needle aspiration biopsy of the intraocular tumour can be performed simultaneously with electrode insertion.Undoubtedly, the size of the intraocular tumour destruction occurred after clinical use of the ECL method will depend not only on the parameters of the procedure but also on the duration of the post-ECL time period. To elucidate these issues, the insightful information from clinical trials will be required.Our experimental study indicates that the new ECL method employing the original combination of electrode placement combines minimal tissue injury and complete tumour destruction under the area of electrode application. The use of surface electrode enables to direct and evoke destruction of the whole tumour base area.The combination of surface and intrastromal electrodes allows the minimal damage to the scleral integrity in the site of tumour base projection. Further experiments dealing with variations in depth of the intrastromal electrode insertion and the amount of current, coupled with bioimpedancemetry, will enable the regulation of morphological changes within the tumour.The proposed ECL method is promising and can be considered as optional for the organ-sparing treatment of large-sized intraocular tumours. Further optimization of the ECL parameters, as well as the development of sets of surface and intrastromal electrodes for different types of tumours, is required.


Background and Objectives. To determine the most appropriate technique for tumour followup in experimental therapeutics, we compared ultrasound (US) and magnetic resonance imaging (MRI) to characterize ectopic and orthotopic colon carcinoma models. Methods. CT26 tumours were implanted subcutaneously (s.c.) in Balb/c mice for the ectopic model or into the caecum for the orthotopic model. Tumours were evaluated by histology, spectrofluorescence, MRI, and US. Results. Histology of CT26 tumour showed homogeneously dispersed cancer cells and blood vessels. The visualization of the vascular network using labelled albumin showed that CT26 tumours were highly vascularized and disorganized. MRI allowed high-resolution and accurate 3D tumour measurements and provided additional anatomical and functional information. Noninvasive US imaging allowed good delineation of tumours despite an hypoechogenic signal. Monitoring of tumour growth with US could be accomplished as early as 5 days after implantation with a shorter acquisition time (<5 min) compared to MRI. Conclusion. MRI and US afforded excellent noninvasive imaging techniques to accurately follow tumour growth of ectopic and orthotopic CT26 tumours. These two techniques can be appropriately used for tumour treatment followup, with a preference for US imaging, due to its short acquisition time and simplicity of use. Colorectal cancer is the third leading cancer in the world in men and second in women with 1.2 million new cases identified in 2008. In terms of mortality, this cancer takes the second place after lung cancer with 608 700 deaths in 2008 [in situ generation or via implantation of tumour fragments into appropriate syngeneic or immunosuppressed hosts. In situ models, including chemically induced, transgenic and spontaneous models, are considered less adapted for the evaluation of experimental therapies because of their inherent variability and the relatively long-time delay required for tumour growth and response to treatment [Murine colon tumour models can be obtained via reatment . While ereatment . Moreovereatment –8.αvβ3) and E-selectin, which are important targets for antiangiogenic therapy [In the present study, we have chosen to develop a murine orthotopic colon carcinoma model that could better reflect the clinical colon carcinoma situation, in order to be able to evaluate new antiangiogenic, antivascular, and antimetastatic approaches. To do so, we selected the colon carcinoma 26 (CT26) model because we have recently observed that this tumour is well vascularized and expresses cellular adhesion molecules, for example, alpha v beta 3 , computed tomography (CT), positron emission tomography (PET), single-photon emission-computed-tomography (SPECT), ultrasound imaging (US), and bioluminescence imaging (BLI) are now being frequently used, and their performance is continuously improving , 11.in vivo studies on small animals [Although these various imaging techniques have been used for  animals –12, the N-nitroso-N-methylurethan [2-humidified atmosphere in Dulbecco's Modified Eagle Medium  containing 10% fetal bovine serum , 100 μM of streptomycin, and 100 U/mL of penicillin. The CT26 cell line was originally obtained from an undifferentiated colon carcinoma chemically induced by lurethan  that waslurethan . This ceStudies were carried out in Balb/c female mice , aged from 6 to 7 weeks. Animal experiments were conducted according to European and national guidelines and were approved by the institutional ethics committee.3. These fragments were evaluated to contain approximately 9 × 105 tumour cells, using hemocytometer measurement after trypsin disaggregation. The tumour fragments were transferred into sterile phosphate buffer saline and inserted subcutaneously using a 12 gauge trocar (38 mm) into the mouse flank previously disinfected with alcohol. For the CT26 line, tumours were implanted every two weeks. In these conditions, the take rate was 100%. A mouse bearing a subcutaneous CT26 tumour was sacrificed, the tumour was resected, placed into DMEM culture medium, and cut into fragments of 20–30 mmAllografts were prepared from ectopic tumours passaged as described previously in In order to evaluate histology and tumour weight evolution of both models, five mice per model and per time point were implanted. At the indicated time points , mice were sacrificed and their tumours resected. A photograph of each tumour was captured using a Canon Digital IXUS 80 IS camera. Finally, the tumour was weighed and flash frozen in liquid nitrogen. Ten-micron frozen tissue sections were obtained in the middle of the tumour and stained with haematoxylin. The orthotopic tumours were compared with a caecum of healthy mice and the ectopic CT26 tumour.μg/mL) for 2 h at 37°C. The sections were next incubated for 30 min with goat anti-rat biotinylated antibody (0.5 mg/mL) and finally with the streptavidin-conjugated peroxidase . The staining was carried out with 3,3′-diaminobenzidine  substrate to obtain a brown precipitate. Tissues were counterstained with Gill's haematoxylin to visualize cell nuclei. Ten-micron frozen tissue sections were placed on Polysine slides . Immunostaining for the evaluation of the vascularity was performed using a three-step procedure as previously described in , 18. BriFor each tumour, immunohistochemistry assessment was obtained in the central slice of the tumour. In this slice, ten representative images were realized and analysed. All stains were performed at the same time and images digitization was made under identical lighting conditions to insure that analyses from different slides were comparable. ex vivo tumour perfusion, mice were injected i.v. with 100 μL of Hoechst 33342 solution  1 min before sacrifice. Tumours were removed and flash frozen in liquid nitrogen. Ten-micron frozen tissue sections were realized in the middle of the tumour and were placed on Polysine slides. Tumour section was incubated 2 h at 37°C with the rat anti-mouse CD31 monoclonal antibody  and further incubated with an anti-rat fluorescein isothiocyanate (FITC) secondary antibody (Sigma) at 1/400 dilution for 45 min in the dark. Slices were mounted in aqueous media  and observed under fluorescence microscopy  equipped with a camera Retiga 2000 . Photographs were realized in the edge (n = 4) and in the centre (n = 4) of the tumour. Intensity of fluorescence emitted by the perfused cells was quantified with ImageJ.In order to assess 2, per tumour, and per time point. For the characterization of the tumour vascularization, we developed a method for the quantification of various markers using the ImageJ software . The imaμL per mouse). A subcutis incision near the tumour for the ectopic model and a laparotomy for orthotopic model were realized in order to visualize the tumour vascularization under a Leica fluoromacroscope (Z6 APO), and the image acquisition was performed with a Leica camera (DFC 340Fx). In our experimental conditions, it was possible to visualize the tumour vascularisation in the depth of 3.1 mm to 0.09 mm.Mice were anaesthetized with a mixture of ketamine/xylazine , and a solution of albumin alexa fluor 594  was injected intravenously (i.v.) into the tail vein . Physiological parameters of temperature and breathing rates were controlled.The MRI studies were performed using an MRI spectrometer 7T PharmaScan  dedicated to small animal experimentation. Images were recorded using a 36 mm inner diameter birdcage Bruker RF probe. Mice were anaesthetized using 1.5% isoflurane gaz with a mixture of air/O2) was multiplied by the thickness of the slices (0.83 mm) to calculate the volume (in mm3) of each slice. To obtain the volume of the tumour, the volume of each slice was summed.In order to determine the tumour volume, Gradient Echo imaging sequence was performed. Thirty axial slices of 0.83 mm thickness with a plane resolution of 0.208 mm were acquired during 3 min, which cover the entire volume of the tumour. For the calculation of the tumour volume, the tumour region was delineated on each section with ImageJ software . The areT2-weighted images of the centre of tumour were obtained thereafter for an accurate depiction of the anatomy and the quantification of necrosis by using a Spin Echo sequence with an echo time TE of 54 ms. These slices have a thickness of 0.6 mm and a plane resolution of 0.123 mm for an acquisition time of 15 min. Hemorrhagic necrosis containing deoxygenated haemoglobin appears mainly in hyposignal. For the calculation of the hyposignal area a threshold of 8% was applied in the images from T2-weighted images. The percentage of hyposignal area was obtained by the mean of the number of pixels under the threshold per slice divided by the slice area.Five axial T2-weighted Spin Echo images were recorded to assess inflammatory oedemas [T2 maps were computed from these multicho SE images by using the following equation :S is the signal intensity obtained at each echo time (TE) and S0 is the magnitude of the signal without relaxation.  oedemas . T2 mapR1 = 1/T1) maps were obtained by using saturation recovery gradient echo images with variable flip angles before and after contrast agent administration [R1 and maximal signal intensity (Smax) were calculated according to Seshadri et al. [R1 of the tumour (ΔRt1) and the muscle (ΔRm1) were performed to calculate the slope and y-intercept value at time zero. The slope represents the permeability of tumour vessels to DOTAREM, and the y-intercept provides a measure of tumour vascular volume [Multislice relaxation rate  coupled with a high-frequency ultrasound probe . The system can reach a frame rate of 20 000 images/s with special resolution of 100 π/6) × L × W × H, where H is the height, L is the length, and W is the width in cm [By using classical ultrasound imaging, the so-called B-mode, the operator localized the tumour within the imaging plane in real time by placing the probe on the anaesthetized mouse. Once the probe was positioned, B-mode acquisitions were performed to obtain a morphological image of the tumour in two perpendicular imaging planes  in order to measure their largest diameters. Thus, three diameters  were recorded, giving access to volume quantification according to the following spheroid formula:  and cellularity. 3 to 13.6 ± 0.5 × 103 cells/mm2 for the ectopic model and from 18.0 ± 1.3 × 103 to 12.6 ± 1.0 × 103 cells/mm2 for the orthotopic model, respectively. After the 11th day until the 18th day the cellularity remained stable for both models. A significant increase in cellularity was however observed for both tumour implantation sites after day 18.Cellularity presented in The vascular organization was observed on both models 15 days after tumour implantation using a 3D evaluation method based on spectrofluorescence. Figures T2*-weighted sequences in which axial slices were positioned in the tumour area . In these axial slices, tumour burden was easily detected without the use of a contrast agent. The tumour mass appeared in grey and the outline was relatively well defined as shown in Figures 3 measured at day 5, to large tumours up to 3000–3400 mm3 measured at day 18, are shown on We next explored the characterization of colon carcinoma CT26 models using MRI. Coronal image was obtained from T2 weighted images provide valuable information on extracellular water such as inflammatory oedemas displaying hypersignal [ersignal  and hemoersignal , 26. T2 value in ectopic and orthotopic tumours (T2 decreased significantly (P < 0.05) from day 5 (56.4 ± 3.0 ms) to day 11 (46.1 ± 1.5 ms) and remained stable beyond this time point . For the orthotopic model a similar profile was observed. The absence of hypersignal indicated that there was no detectable inflammatory oedema from day 11 to day 18 [T2 value on day 5 could indicate a slight inflammation due to tumour implantation.In a first place, we measured the global mean  tumours . For theo day 18 , 26. TheIn a second place, the percentage of hyposignal was calculated. The fraction of hyposignal (R1 in the tumour (ΔRt1) and the ΔR1 of the muscle (ΔRm1) are represented in Rt1/ΔRm1 ratio on the edge of the tumour was higher than the ΔRt1/ΔRm1 ratio calculated in the centre of the tumour  slope was slightly steeper as compared to the ΔRt1/ΔRm1 (edge) could also suggest a higher vascular permeability in the centre of the tumour.The DCE MRI experiments provided quantitative data on the tumour perfusion changes Figures .  Figuree tumour , showingMoreover, the intravenous injection of a chromophor compound 1 min before sacrifice provided convergent histological data about tumour perfusion Figures . If we eex vivo fluorescent data and in vivo MRI data are consistent with a lower perfusion rate in the centre of the tumour.Overall, We also evaluated echography as a noninvasive US imaging technique and assessed its usefulness for the monitoring of colon CT26 growth in mice. As presented in 3 for the ectopic site  to late growth stage (day 21). The anatomical resolution allowed the precise determination of 3D tumour volume in both models. This volume was correlated to  tumours . Moreove tumours . In addid oedema . Howeverex vivo fluorescent study. Therefore, perfusion experiments could be relevant to assess antivascular activity in vivo of vascular disrupting agents. Indeed, the effect of antivascular disrupting agents will affect the haemorrhagic zone often located in the centre of tumours and induce the formation of a viable rim which could then be measurable in a noninvasive way. The DCE MRI experiments provided to quantify perfusion changes in tumour. The results obtained in this study are in good agreement with histological results previously reported . The higMoreover, thanks to magnetic resonance elastography (MRE), the viscoelastic properties of the tissue can be obtained to better characterise the tumour , 34. In As described above, MRI presents several advantages and is particularly adapted to follow tumour growth and tumour stage during treatment. However, three critical points should be considered: MRI remains an expensive technique, the operator should be qualified, and this modality could take from 5 to 20 min acquisition time per mouse .Concerning the results obtained using ultrasound imaging, data provided a good estimation of tumour volume in both implantation models, as expected . MoreoveTumour blood flow could be assessed by ultrasound imaging without the use of contrast agent by using appropriate probes and Doppler mode. Unfortunately, in our study, we could not detect blood flow in the tumour centre with the 15 MHz probe used. This is probably due to the low sensitivity of the ultrasound device (2 m/s) that could not detect such small vessels. The lack of visualization of tumour microvessels is also probably due to a lower venous pressure which causes flow stasis . It has Tumour elastic properties could also be measured with US imaging. As a matter of fact, shear wave elastography has also been developed and is now used in the clinic for breast tumour grade determination , 40. We Finally, US imaging was found to be easy to perform and required only 1 to 5 min per mouse. This technique can advantageously be applied to the assessment of longitudinal CT26 tumour growth in the context of antitumour treatment cf. .In this study, non-invasive clinically available imaging techniques were evaluated to detect and follow up the growth of CT26 colon tumour. MRI and ultrasound techniques were shown to be particularly suitable for the accurate follow up of CT26 colon carcinoma growth in both ectopic and orthotopic mouse models. These imaging techniques and their pertinent parameters related to tumour growth, haemorrhagic areas, and perfusion could be assessed on CT26 mouse models. This first set of information is very important to define valuable imaging parameters that could be helpful for the diagnosis of tumour grade in the clinic and also for the monitoring of new antitumour approaches.The first figure in Supplementary Material: “Method used for the calculation of tumour volume by MRI. Images show an example from an ectopic tumour on day 15 using a coronal axe (A), and an axial view .”The second figure in Supplementary Material: “Ultrasound imaging of CT26 ectopic tumour. Axial plan (A), sagittal plan (B), coronal plan (C) and 3D view (D). Acquire with 8 MHz probe ”.Click here for additional data file.


As a result of advanced treatment techniques, requiring precise target definitions, a need for more accurate delineation of the Clinical Target Volume (CTV) has arisen. Mathematical modelling is found to be a powerful tool to provide fairly accurate predictions for the Microscopic Extension (ME) of a tumour to be incorporated in a CTV. In general terms, biomathematical models based on a sequence of observations or development of a hypothesis assume some links between biological mechanisms involved in cancer development and progression to provide quantitative or qualitative measures of tumour behaviour as well as tumour response to treatment. Generally, two approaches are taken: deterministic and stochastic modelling. In this paper, recent mathematical models, including deterministic and stochastic methods, are reviewed and critically compared. It is concluded that stochastic models are more promising to provide a realistic description of cancer tumour behaviour due to being intrinsically probabilistic as well as discrete, which enables incorporation of patient-specific biomedical data such as tumour heterogeneity and anatomical boundaries. Advanced radiotherapy techniques like 3D Conformal Radiotherapy (3D-CRT), Intensity-Modulated Radiation Therapy (IMRT), and Image-guided Radiation Therapy (IGRT) restrict the high dose region to defined target volumes to spare adjacent normal tissue. The margins are generally reduced for modern radiotherapy techniques due to (a) more accurate organ specification with the use of daily image guidance that results in minimization of set up error, and (b) superior conformity of dose distribution to irradiation target volumes. However, a successful implementation of these techniques, that is, achieving an acceptable Tumour Control Probability (TCP) and Normal Tissue Complication Probability (NTCP), requires very accurate target volume delineation. According to ICRU report 50, the “Clinical Target Volume (CTV) is a volume encompassing visible Gross Tumour Volume (GTV) and subclinical malignant disease” . Since sen(D)−, where n(D) is the expected number of surviving clonogens.) Therefore, in order to confidently reduce the irradiated target volume, as is the trend with current treatment techniques, the pattern of microscopic extension needs to be known or predicted.Among radiotherapy target volumes, delineation of the Clinical Target Volume (CTV) is the most controversial. To date, there is no consensus regarding the extent of histological disease, thus the question of how far CTV is extended beyond GTV is mostly left to the discretion of radiation oncologists based on their experience, depending on patient's histopathological data. The uncertainty in CTV represents a limitation on reduction of the irradiated target volume. When the irradiated target volume is reduced due to dose conformity of new treatment modalities, NTCP is improved. On the other hand, the issue of CTV fuzziness becomes a cause of concern because any PTV reduction enhances the risk of missing a part or a few cells of subclinical disease, as illustrated in avascular phase and vascular phase and transition between these two phases requires angiogenesis, a process which involves development and recruitment of blood vessels to supply tumour cells with nutrients [Normal growth and regeneration of an organ requires cells to undergo cell division and to proliferate. The rate of proliferation, however, is systematically regulated to ensure the balance between cell proliferation and cell loss as well as integrity and functionality of each organ. This regulation occurs at cell cycle check points where progression to a subsequent phase is prevented unless prerequisites are satisfied. DNA lesions are recognized at check points that lead onto repair pathways . Normallutrients , 6. Tumoutrients . haptotaxis [The Extracellular Matrix (ECM) is the external part of tissue on which cells reside. It provides structural support to the cells, regulates intercellular communications and so forth. The ECM also imposes spatial constraint on tumour proliferation. On the other hand, the tumour invasion is known to be facilitated by gradients in the ECM density . These gradients cause the cells in the outer layer of a tumour to break away from the primary tumour mass and move along the gradient, a phenomenon called ptotaxis . It is kptotaxis . Apart from proliferation and haptotaxis, other factors like cell-cell adhesion, cell-matrix adhesion, and ECM density also affect cell motility in the course of tumour invasion .In summary, tumour evolution is an interrelated multistage process that starts from a series of cancer-associated gene mutations leading to formation of a colony that could further invade adjacent tissues and finally metastasize in distant organs. Better understanding of biological mechanisms of cancer development helps to anticipate the behaviour of the tumour that undoubtedly leads to a better treatment efficacy.Mathematical modelling is a suitable tool to generate algorithms to correlate information acquired from imaging techniques to the pattern of growth and tumour invasion. In a typical course of model development, biological phenomena are represented in mathematical equations. The solutions of the equations, in return, provide predictions of tumour evolution, tumour aggressiveness in a given patient, and so forth. The validity of a model is then examined by comparison with available actual data, and iteration is performed until an adequate match is reached and thus a plausible model is obtained. A semirealistic model developed in this manner provides an insight into biological mechanisms of tumour growth and invasion under a variety of circumstances. It also allows for assessment of potential treatment regimens. The model could be useful for clinicians in clinical tumour volume definition.Oncogenesis can be modelled at three levels: (1) sub-cellular level, (2) cellular and microscopic level that concerns individual cell behaviour while taking into account cell-extracellular matrix (ECM) interactions, and (3) macroscopic level that is related to the evolution of tumour in terms of cell density and mostly is based upon reaction-diffusion equations .In this paper, some of the recent computational and mathematical models developed for tumour growth and invasion are reviewed. Two approaches used for modelling, analytical and stochastic, are discussed individually in the following sections.c denotes the density of tumour at location x and time t, ∇·J is the diffusion component , and ρc is proliferation component , where ρ is the proliferation coefficient. Using the Fick's first law that assumes the diffusive flux flows from high-concentration regions to low-concentration regions, the diffusion component is related to tumour cell density as follows:Analytical modelling of tumour growth has been typically done based on the reaction-diffusion equations in the literature. Swanson et al.  reviewedD is diffusion coefficient representing active motility of cancer cells and ∇ denotes spatial gradient operator. The first term, the diffusion component, is related to the periphery of the tumour while the second term, the proliferation component, pertains to active part of tumour core and is described by cellular proliferation laws  [D, is constant throughout the brain.Brain tissue is homogeneous thus diffusion coefficient, ρc is constant.Tumour growth is generally exponential thus c = f(x), wheref(x) is initial profile of the tumour and there is no migration beyond brain boundaries.Boundary condition: Thus  takes th growth) . The assThus  reduces c, is a function of the ratio of ρ/D thus two different tumours whose different combinations of ρ and D result in the same ratio of ρ/D, appear the same at a single observation time. Hence, just a single MRI/CT image is not sufficient to estimate CTV correctly without knowing the pattern of tumour cell density distribution. One of the consequences of  is that (i)D, is not uniform and is a function of location in the brain tissue.Complex geometry of brain is introduced, thus diffusion coefficient, (ii)D being a function of x as follows:Equation  is appliA more realistic approach was taken by Swanson et al. , 17who iνG and νW, respectively. Fisher's approximation (D = ν2/4ρ) was then applied to correlate velocity, ν, of detectable tumour margin with proliferation rate and diffusion coefficients. According to CT scans of the right hemisphere (predominantly grey matter), νG was identified to be 0.008 cm/day, thus Fisher approximation gives DG = 0.0013 cm2/day, and DW being almost five times of DG becomes 0.0065 cm2/day. To assign diffusion coefficients to corresponding brain cells, spatial distribution of white and grey matter was adopted from the brain web database [2. This means that any part of tumour having a concentration below this threshold is not detectable on a MRI image. The comparison between detectable part and simulated profile provides an insight into how far and at what concentration microscopic disease is invaded beyond visible tumour. This model that derived the behaviour of glioma according to two factors (“D” and “ρ”) demonstrates that the distribution of ME in invasive gliomas does not follow an isotropic pattern that is invariably assumed by clinicians for definition of CTV.To determine the model parameters, 12 serial CT scans of a patient, diagnosed with astrocytoma, during his terminal year were examined to derive estimations for velocities of tumour margin advance through grey and white matter, database . ApplyinThe biomathematical modelling based on  in conjuin vitro experiments on the three dimensional pattern of growth of GBM spheroids. It was concluded that GBM spheroids consist of two classes of cells, namely, proliferating core cells and peripheral migrating cells. This finding was later included in other models like the model of Thalhauser et al. [The evolution of mathematical modelling to gain insight into the mechanism of GBM growth and invasion initiated by Swanson et al. , 17 was r et al.  in whichr et al.  incorporg(c) is the cell loss due to chemotherapy and defined ask1 and k2 are positive constants. The spatial-temporal evolution of the brain tumour in the presence of chemotherapy was investigated by Tracqui et al. , 12. Twef(c), is typically taken as a linear function of c  or a nonlinear function of c (logistic proliferation) when the proliferation is limited, since cell density is close to its maximum:The proliferation term,c2 present which is resistant to the first course of chemotherapy but sensitive to the second course. The insensitivity of the second population was considered to be due to mutations from the radiotherapy administered three years earlier. Given this condition, the system was described mathematically as follows:ρ1 and ρ2 are proliferation rates corresponding to the first and second cell density, respectively, and variable c represents the total density of tumour cells (c = c1 + c2). The area of tumour was evaluated at each successive CT scan and then the data was compared to the values derived from . The comD = 1.2 × 10−7 cm2/s which is comparable with estimation of glioma cell migration rate obtained from in vitro experiments [After optimization and identification of unknown parameters, the identified values were found to be in agreement with known biological data .Woodward et al. , 34 modig(c) to be a periodic function such that for the time periods chemotherapy is on, g(c) is equal to a specific positive constant, k (indicating the rate of cell loss due to chemotherapy), and otherwise is zero. The model was originally formulated assuming homogeneous drug delivery and further developed to take into account heterogeneity in drug delivery, whereby drug delivery is expected to be less in white matter compared to that in gray matter. The experimental observation of shrinkage of gliomas in specific areas together with persistent growth in other areas of the brain following chemotherapy was explained by this model. Swanson et al. , 35 inveClatz et al.  developeBondiau et al.  applied D and k denote the dose and tumour carrying capacity, respectively. R is the probability of death of cancer cells −) due to radiotherapy.The effect of external beam radiation therapy was incorporated in the reaction-diffusion model in the study of Rockne et al. . Therefou, ρ, and c were designated for mechanical displacement of cell-ECM composite, density of ECM, and cell density, respectively. The parameter r denotes the proliferation rate of cancer cells. Thus the reaction-diffusion formula (cell conservation equation) takes the bllowing form:In previous models, passive translocation of cells due to ECM-cell interactions and active cell migration were overlooked. Retaining reaction-diffusion formula as the framework, Tracqui  introducu/∂t. Equation  and G denote the rate of formation and loss of ECM, respectively. For the sake of simplicity, ECM turnover was neglected, that is, S = G = 0. Thus u/∂t. EquD1 (assuming constant ECM density), and haptotactic response to the ECM gradient. As a matter of fact, cancer cell motility depends on both ECM gradient and density, thus this was a simplifying assumption. The series of differential equations constituting the model are as follows:c, v, m denote the cancer cell density, the ECM density and the concentration of Matrix Degrading Enzyme (MDE), respectively. The parameters ϑ1 and ϑ2 are fractions of unit volume occupied by cancer cells and ECM, respectively. μ1, μ2, γ, D3, α and λ denote proliferation rate of cancer cells, remodelling rate of ECM, degradation rate of ECM, MDE diffusion coefficient, the rate of release, and removal of MDE, respectively. Finally, χ is designated for haptotactic function. Equation  providen), ECM (f), MDE (m), and oxygen (c). The interaction of these parameters was represented in a set of differential equations, as follows:Such a hybrid model of tumour growth and invasion was developed by Anderson . The forγ and α, respectively. The hybrid model, that follows the path of each individual cell, requires discretising the system of differential equation based on finite difference method in a given time and space steps [i and j represent the location and q specifies the time. The coefficients P0, P1,…, P4 are probabilities of transition from the grid in question to the respective neighbouring grids. Unlike purely continuum modelling, the hybrid model, being intrinsically multiscale, allows for investigation of the effect of tumour cell heterogeneity on the morphology and phenotypic diversity of invading vascular tumours  [in vivo and in vitro experiments to examine the role of harsh environment  in the invasive morphology of tumours [As indicated in the first line of , oxygen ce steps . Each poe cells) . In the e cells) , 52, the tumours , 53. TheMalignant tumour invasion, driven by haptotaxis, both in the form of travelling waves (continuum models) –56 and hin silico prediction of malignant progression of tumour corresponded well with imaging (MRI) and histologic data of three GBM patients who had approximately similar size of tumour but different hypoxic and necrotic ratios on their MR images. In the context of microscopic extension, this model can predict local invasion. However, it cannot visualize those microscopic clusters of cells detached from main mass of tumour, since it overlooks migration (via haptotaxis).The most recent work in the continuum deterministic framework is the study of Swanson . In thisAnalytical modelling based on conservation of cells has evolved from basic models such as the one proposed by Murray's group –14 to veStochastic models are guided by probability distribution. The various techniques used in stochastic modelling are dominated by Monte Carlo and Markov approaches which are generally employed in the simulation of biological systems.Xis are random variables having Markov property. A Markov chain is the simplest Markov model which is a chainlike random process that transforms from one state (i) to another (j) by a transition matrix whose elements are described asMarkov models are stochastic models which simulate the state of systems with time-dependent random variables possessing Markov property. A stochastic process has Markov property (or memoryless property), if the probability distribution of future states depends only on the present state and not on the preceding sequence of events. This reads mathematically asBenson et al.  producedMonte Carlo (MC) models are widely used in the field of cancer biology and treatment since this method is particularly useful for simulating systems with considerable uncertainty in parameters.in vivo cancer development, Qi [V = V0exp(A/B(1 − exp(1 − Bt)), where V is the volume of tumour at time t and V0 is the initial volume. A and B are parameters). Smolle and Stettner [in vivo brain tumour growth model which was developed by Wasserman et al. [The earliest developed MC models of tumour growth date back to early 80's, for example the work of Duchting and vogelsaenger  for smalment, Qi  simulatement, Qi  later adStettner  considerStettner . Aimed tn et al. . The modin vitro study conducted by Deisboeck et al. [One of the common approaches in stochastic modelling is the Cellular Automaton (CA) method which employs a grid lattice, with each site in the grid accommodating a finite number of cells in specific states, to grow a tumour from a few cells to macroscopic stages. When the time is incremented by one, the defined biological rules determine the updated states of cells in terms of their current states and microenvironment. A 3D cellular automaton model of untreated brain tumour was developed by Kansal et al. , 70. Thek et al.  and was k et al. , wherebyk et al. . In the k et al. . in vivo stochastic model was developed by Stamatakos et al. [G0 cell regions and dead cell regions. The metabolic state of each GC was determined depending on the distribution of its contained cells in different phases. The initial NBC distribution is derived from imaging and histopathological data of each individual patient, whereby the tumour region is apportioned to three metabolic layers: proliferating, resting, and necrotic. Time is discretized and at the end of each time step the GC mesh is updated such that transitions between different metabolic states are estimated and applied . The time was incremented at the end of each scan and the process iterated. In order to investigate the radiotherapy effect on tumour shrinkage, the Linear Quadratic (LQ) model of surviving fraction (S = eαD−βD2−) is employed. Three sets of radio sensitivity parameters (α and β) were assumed corresponding to proliferative, necrotic, and resting states and the tumour regression was simulated for three specific cases: standard fractionation/radiosensitive tumour, standard fractionation/moderately radiosensitive, and hyper fractionation scheme/radiosensitive tumour [Aimed to simulate untreated tumour growth and also the response of tumour to different schemes of radiotherapy, a four dimensional, patient-specific, s et al. , 74, 75.s et al. , as illue tumour . The simin vivo tumour growth and response to radiation. In the same framework, Dionysiou et al. [The model was gradually improved to take into account possible parameters involved in tumour growth and response to radiotherapy to achieve a more biologically realistic description of cancer biology and treatment. Antipas et al.  studied u et al. , 79 condu et al. , 74 and u et al. , 77–79, −1 cell cycle). The emergence of glycolytic phenotype associated with anaerobic metabolism pathway of cells was investigated in subsequent extension of the model [Individual-Based Modelling (IBM), which has gained popularity for modelling of biological processes, is another class of stochastic modelling . In IBM he model , and morhe model . The effhe model , 83 and he model , 51. To summarize, in clinical situations, physicians propose CTVs based on their experience of the extent of malignant tumours growth. Therefore, the ability to accurately model the tumour extension at microscopic scale is highly desirable. Within the realm of stochastic modelling, a significant number of research works has been developed to contribute to the understanding of the tumour growth and invasion via a variety of classes of Monte Carlo models. However, irrespective of the class, these studies aim to gain insight into either the biology of cancer growth in general terms or the response of tumour to radiotherapy rather than the microscopic extension of tumour which is to be incorporated in CTV. Hence, there is room for investigation in this respect, in the light of information acquired from these studies. An infiltrating neoplasm undergoes several stages in the course of its growth and progression and understanding of the mechanisms governing the evolution of tumour is required to deliver an appropriate therapy which results in optimal tumour control and reduced normal tissue side effects. Mathematical modelling is recognized as a great tool to facilitate this understanding. Furthermore, mathematical models provide predictions of the probable response of tumour to therapeutic regimens in a variety of circumstances, different in terms of factors such as the tumour microenvironment, and stage. In this paper, we have reviewed the evolution of mathematical modelling of tumour growth and invasion in both analytical and stochastic approaches. Analytical models are capable to describe the behaviour of tumour at macroscopic level for specific conditions; however, they fail to provide predictions at microscopic (cellular and subcellular) level. In addition, the ongoing research to enhance the limited insight into complex and dynamic cancer systems may reveal some further parameters which have to be included in models. However, analytical models are not flexible for these modifications. On the other hand, stochastic models efficiently depict the characteristic and behaviour of tumour as this class of modelling enables introducing new parameters as well as specific anatomical boundaries. Finally, we came to believe that while none of the above-mentioned models address explicitly the microscopic extension of tumour, they have the potential to be used to deduce the extent of subclinical disease which is not detected by imaging techniques. To serve this purpose, however, models have to be further modified, applying the relevant biological parameters, to become site-specific. The tumour sites that have a relatively high histopathological data available, such as prostate and gliomas can be potentially modelled and validated faster than those having little or no clinical data related to their microscopic extension.


Tasquinimod is a small molecule with pleiotropic effects on the tumour microenvironment. Tasquinimod inhibits the growth and metastasis of tumour cells in vitro and in vivo. It targets the tumour microenvironment, enhancing the host immune response and inhibiting the angiogenic response. Tasquinimod influences infiltrating myeloid cells in the tumour milieu shifting the balance towards a less immunosuppressive phenotype. Myeloid-derived suppressor cells and tumour-associated macrophages are major components of the immunosuppressive microenvironment and as a result promote tumour growth and favour angiogenesis and metastasis formation. Growing evidence indicates that tasquinimod targets these myeloid cells and modulates local tumour immunity by blocking the interaction between the multifunctional protein S100A9 and its ligands receptor of advanced glycation end products and Toll-like receptor 4. Its anti-angiogenic effects are achieved at least in part through these effects on regulatory myeloid cells and also potentially through inactivating histone deacetylase-4 and reducing expression of hypoxia-inducible factor 1-controlled genes. The aim is to comprehensively review the mode of action of tasquinimod as a novel oral anti-cancer agent. Based on its unique combination of effects, tasquinimod is a novel agent with clinical therapeutic potential in various solid tumours, both alone and as part of rational combination therapy. The tumour microenvironment plays a key role in supporting the growth, invasion and metastasis of malignant tumour cells and in protecting the cancer cells from the host immune response . StromalTasquinimod is a novel oral quinoline-3-carboxamide derivative with multiple effects on the tumour microenvironment, which is currently at an advanced stage of clinical evaluation as an anti-cancer agent. Among other clinical trials, tasquinimod is currently being studied in a Phase III trial in metastatic prostate cancer and in a Phase II trial in hepatocellular, ovarian, gastric and renal cell carcinomas . The aim of this review is to summarise the available data and provide an overview of the biological properties of tasquinimod that contribute to its anti-tumour effects.Immunotherapy for cancer uses the potential of the host immune response to recognise and eliminate tumour cells. Modulation of the immune response has been the subject of intensive preclinical and clinical research to control tumour growth. The innate and adaptive immune systems can mediate anti-tumour immunity; however, as tumours progress, they escape from immune surveillance through various mechanisms . Among tThe association between chronic inflammation and cancer is well recognised; prolonged presence of an inflammatory milieu predisposes to an increased risk for developing cancer and facilitates tumour development and progression , 8, 9. CTAM associated with tumours are predominantly the M2 phenotype, which suppress adaptive immunity, encourage angiogenesis and support metastasis through the expression of cytokines, growth factors and matrix metalloproteases , in contSuppression of tumour-specific T cell sensitisation is an important mechanism that results in uncontrolled proliferation of cancer cells. MDSC present in the tumour microenvironment have the capacity to suppress the cytotoxic effects of NK cells and the adaptive immune response mediated by CD4+ and CD8+ T cells, strongly inhibiting the anti-tumour effects of these cells , 15. HenThe term angiogenesis describes the development and growth of new capillary blood vessels from existing vessels, and this is an essential step necessary to supply oxygen and nutrients to tumours once they are larger than a few millimetres. It has long been recognised that blockade of angiogenesis is a potential anti-cancer intervention, since solid tumours are dependent on this process for sustained growth and metastasis . Hence, Angiogenesis is a complex process regulated by the highly coordinated function of various proteins with pro- and anti-angiogenic functions , 19. As A series of quinoline-3-carboxamide analogues were identified in the early 1980s, and the lead molecule, linomide, was found to stimulate the immune response and inhibit the growth and metastasis of cancer cells in preclinical models. This compound was evaluated in clinical trials, but development was stopped because of unacceptable toxicity, attributed to dose-dependent pro-inflammatory reactions . Tasquinp < 0.05) [The anti-tumour effects of tasquinimod have been documented in a wide range of preclinical models including both rodent tumours and human tumour xenografts Table  22, 24,,24, 25. In addition to inhibiting the growth of primary tumours, tasquinimod prevents metastasis formation in animal models. In a preclinical study of castration-resistant prostate tumour xenografts in mice, tasquinimod inhibited the formation of lung and lymph node metastases and suppressed the formation of tumours in bone after intratibial implantation .Extensive studies have been undertaken to evaluate the mechanism underlying these anti-tumour effects of tasquinimod, focusing on its effects on the immune system and angiogenesis.Infiltrating myeloid cells in the tumour microenvironment are essential for tumour growth, metastasis and angiogenesis , 13, 15.S100A9 is a multifunctional protein which is often co-expressed with S100A8, another member of the S100 protein family. They are classified as damage-associated molecular pattern (DAMP) molecules that are secreted from myeloid cells upon activation . S100A9 High levels of S100A9 have been found in the microenvironment of several forms of tumours, and a high expression level has been correlated with poor tumour differentiation , 38. In Tasquinimod binds to S100A9 and inhibits its interaction with receptors such as RAGE and TLR4. The importance of these interactions in tumour development was illustrated in experiments in knock-out mice showing that in the absence of S100A9 or TLR4, the development of spontaneous prostate cancer tumours was delayed . In the + T cells in the mouse B16 melanoma model [+Ly6Chigh MDSC and increased the subpopulation of CD11b+Ly6G+ MDSC, thus shifting the infiltrating myeloid cell population towards a more granulocytic phenotype [+ tumour-infiltrating macrophages from a strong dominance of M2 into a less suppressive M1 myeloid population (unpublished data). Thus, tasquinimod may alter the balance between M1 and M2 macrophages in the tumour microenvironment further promoting anti-tumour effects.Tasquinimod has shown anti-tumour effects in several other syngeneic tumour models, possibly by modulating the accumulation and function of regulatory myeloid cells, thereby reducing immunosuppression in the tumour microenvironment. Accordingly, the combination of tasquinimod with immunotherapy using tumour-targeted superantigens (TTS) resulted in a significant enhancement of anti-tumour effects and an increase in the number of tumour-infiltrating CD8ma model . It is lhenotype . Tasquinhenotype . FurtherThere are many links between the immune system and angiogenic pathways, including the potential for TAM and MDSC to promote angiogenesis through production of pro-inflammatory cytokines and endothelial growth factors , 13, 43.Tasquinimod has been shown to inhibit angiogenesis in a variety of models: it inhibits endothelial cell growth and capillary tube formation from aortic rings in in vitro (ex vivo) models, decreases the density of tumour microvessels (CD31-positive) and reduces real-time tumour blood flow and tumour oxygenation in vivo . HoweverSeveral mechanisms may underlie the inhibitory effects of tasquinimod on the angiogenic switch. As already discussed, the inhibitory effects of tasquinimod on myeloid cells (MDSC and TAM) also have the potential to inhibit angiogenesis. A recent original research report indicates that another possible mechanism for the effect of tasquinimod on HIF-1-controlled genes may be interaction with histone deacetylases (HDAC). Histone modifications such as acetylation are involved in chromatin alterations and tumorigenesis . HDAC fu+M2 TAM in two mouse models [Tasquinimod also increases tumour levels of TSP-1 , an endoe models , 54, 55.The tumour microenvironment promotes the growth and spread of many types of solid tumours through suppression of immune responses and offers an important opportunity for therapeutic intervention, as already discussed. Targeting the myeloid cell compartment is a novel approach that has a vast potential for treatment for many different forms of cancer. Tasquinimod is a leading drug that demonstrated potent effects on modulating the tumour cell microenvironment in several preclinical models leading to extensive clinical evaluations in various types of tumours including prostate cancer. A proof of concept study is ongoing to investigate the clinical activity of tasquinimod in other tumour types, including several where there is a rationale to target MDSC and in which there is already evidence of benefit with anti-angiogenic therapy: advanced or metastatic hepatocellular carcinoma, ovarian carcinoma, renal cell carcinoma and gastric carcinoma after progression with standard therapies (NCT01743469). This trial follows a novel design in which the endpoint is the proportion of patients who have neither progressed (RECIST and Choi criteria ) nor dieTasquinimod has already shown promising effects in the treatment of prostate cancer in clinical studies. There is also encouraging preclinical evidence that tasquinimod can enhance the effects of other anti-neoplastic treatments, including radiotherapy, chemotherapy, androgen ablation and immunotherapy. Considering that inhibitory effects of MDSC on innate anti-tumour immunity represent a significant barrier to cancer immunotherapy, there emerged a strong rationale for combining tasquinimod with other immunological approaches, supported by preclinical work discussed earlier including the combination with immunotherapy using TTS and with a survivin cancer vaccine , 42, 55.Tasquinimod significantly increased the effects of fractionated radiation on endothelial and prostate cancer cells in vitro and in vivo . Based oPreclinical results indicate that the anti-cancer effects of tasquinimod are enhanced when it is combined with taxanes , and a PThe combination of tasquinimod with androgen ablation is also effective in animal models . The synThere is also the potential to combine tasquinimod with newer treatments for metastatic CRPC that address the continued dependence on androgen signalling such as abiraterone, an androgen synthesis inhibitor, and enzalutamide, a potent AR antagonist. The need for more treatment options is highlighted by the recent report that only very modest responses to abiraterone were seen in tumours escaping enzalutamide treatment ; given iTasquinimod targets the tumour microenvironment with the potential to overcome tumour-associated immunosuppression and inhibit angiogenesis, metastasis and tumour growth Fig. . It bindTasquinimod is a promising first-in-class anti-cancer agent with a unique combination of effects on the tumour microenvironment which is currently undergoing clinical evaluation as an oral once-daily cancer treatment.p < 0.001), and median progression-free survival was increased from 3.3 to 7.6 months (p = 0.0042), reflecting a significant delay in disease progression with tasquinimod treatment [Metastatic CRPC is an area of significant unmet clinical need and the initial focus for the clinical development of tasquinimod. In a randomised, Phase II, double-blind, placebo-controlled trial in men with minimally symptomatic metastatic CRPC, 69 % of tasquinimod-treated patients were progression-free at 6 months compared with 37 % of the placebo group (reatment . FurtherIn summary, based on its mechanism of action, tasquinimod represents a novel targeted therapy with potential in many tumour types, and proof-of-concept trials are ongoing; it may also be suitable for combination strategies in different solid tumours. Further preclinical and clinical testing will fully exploit its therapeutic potential as a novel agent targeting the tumour microenvironment rather than tumour cells directly.


Optimal clinical approaches to the treatment of large intraocular lesions located in the posterior pole of the eye remain controversial. In our opinion, the introduction of innovative techniques into the clinical practice of vitreoretinal surgery professionals as well as the implementation of new techniques designed to destroy tumour tissue will make endoresection one of the most promising alternative approaches to choroidal melanoma management, providing an encouraging high-potential eye-saving treatment option in the future. However, in view of a small number of treated cases and a short follow-up period, further research studies are required to evaluate the effectiveness of the electrochemical lysis procedure during endoresection. Choroidal melanoma (CM) is characterised by an extremely poor prognosis, endangering both the visual functions in patients and their lives, due to a high risk of metastatic disease 3%–16%) [%–16% 3,, 14. In The range of organ-saving techniques available for the treatment of CMs located in the posterior pole of the eye is rather limited: transpupillary thermotherapy (TTT), transscleral thermotherapy, photodynamic therapy (PDT), radiation therapy, and brachytherapy.Attempts to modify commonly accepted parameters of the techniques  used in large CM treatment, with the purpose of increasing their therapeutic effect, generally result in a higher number of complications, both intraoperative and postoperative , 6, 8, 9With this in mind, we decided to take a new look at endoresection as an alternative organ-saving technique of surgical treatment of large CMs.This technique, as it has been described in a number of studies , 13, 15,The objective is to develop an innovative combined approach to endoresection of large intraocular tumours localised in the posterior eye pole, involving intraocular electrochemical lysis (ECL) at the stage of tumour destruction.3N0M0)  received endoresection coupled with intraocular ECL. The average age of the patients was 55.4 years.Three patients (three eyes) with large CMs  with an electric charge of 20–25 coulombs. During the ECL procedure, an innovative technique of combined positioning of two electrodes was used, with one surface  electrode serving as the anode, and the other (intraocular) electrode serving as the cathode. The extrascleral electrode was made in the form of a platinum wire mesh (wire diameter of 0.05 mm and mesh size of 0.1 mm). The electrode diameter was 3 mm. The lower surface of the electrode was embedded into an insulating material, while its upper surface remained exposed. The electrode had a handle in the form of an angulated spatula that allowed extrascleral manipulations to be performed to guide the electrode into the area of the tumour basal projection, suitable for further moving it along the scleral surface within the tumour outline. The intraocular electrode was made in the form of a 23 G platinum needle with a curved intratumoural tip. With the exception of its intratumoural section, the electrode was coated with biologically inactive dielectric material .Before starting endoresection, phacoemulsification was done through a scleral incision. Following this, a 23 G three-port vitrectomy was performed by removing the posterior hyaloid membrane, and restrictive endolaser coagulation (λ = 532 nm) was done in three coagulate rows around the tumour at a distance of 2–3 mm from the visible edge. It was followed by retinectomy along the coagulated border, the retina was separated off, and the tumour was fully exposed. Laser coagulation of the choroid (λ = 810 nm) in continuous circular-wave mode was performed at 1 mm from the retinectomy borders. The next step included air–fluid exchange. Subsequently, an endoilluminating device (27 or 29 G) was positioned transsclerally at 4.4 mm from the limbus.The incision of the conjunctive and Tenon’s membrane was made to insert and position the electrode (anode) extrasclerally within the most suitable tumour meridian at 5–6 mm from the limbus. Using the spatula, a tunnel was created and, with the help of the handle, the electrode was guided through it to the area of the tumour projection on the sclera, so that a close contact between the electrode and the sclera could be maintained. To control the proper electrode positioning, sclerocompression was used. An assistant surgeon was holding the handle tightly to keep the electrode in place.A pars plana sclerotomy was performed at 3.5 mm from the limbus in the quadrant that allowed the best possible access to the tumour, and the intraocular electrode (cathode) was inserted intravitreally . The intOnce the extrascleral and intraocular electrodes were in place, the ECL procedure was performed at the amperage of 20–25 mA within a period, which was required to decompose the tumour around the cathode; the decomposition process was accompanied by the formation of liquid cell debris that was removed during the ECL procedure using a vitreous cutter  and 5. AZ) were automatically recorded, and the software application was plotting the real-time tissue impedance curve. Stable impedance readings with minimal fluctuations (Z) served as a prognostic criterion for tumour necrosis.The effectiveness of the ECL procedure was evaluated by means of bioimpedancemetry—the process of measuring malignant tissue impedance within the area between the electrodes when it was exposed to alternating variable frequency electric current. Multiple changes in lysed tissue impedance were measured using a pilot unit at frequencies of 2 and 10 kHz; to obtain the impedance values, the ECL procedure was interrupted for 1 or 2 s at 10-s intervals. Bioimpedancemetry was performed using the same electrodes as for the ECL procedures. Impedance values .As to long-term postoperative results (1.5–3 years after the surgery), ocular fundus examination in all cases showed the surgical coloboma of the choroid located at the site of the excised intraocular neoplasm, with no signs of pigmentation over the entire scleral bed or around the periphery. Neoplasm recurrence or remote metastasis was not found in any case.In the late postoperative period, one patient who had previously suffered from 10-mm thick CM was found to have recurrent retinal detachment in the silicone-oil-filled eye due to the rupture over one of the retinopexia edges, which was associated with progressive proliferative vitreoretinopathy. The patient did not receive any repeated surgical treatment.In our opinion, endoresection of large intraocular tumours with posterior location suggests total tumour removal within the boundaries of the intact tissues, as distinguished from other organ-saving techniques that cause destruction and absorption of necrotised tissue due to therapeutic exposure with subsequent formation of conjunctive chorioretinal scars; these scars can retain viable tumour cells and become a focus for neoplasm recurrence with poor prognosis for repeated treatment options. Endoresection disadvantages include the risk of viable tumour cell dissemination, technical problems associated with the neoplasm removal, haemorrhage, and retinal detachment. Our new endoresection technique has been developed on the basis of the experience and results reported by the authors , 13, 15 At present, endoresection is commonly used after irradiation therapy, brachytherapy, TTT, or PDT. In our opinion, however, this approach is associated with a number of technical problems due to the scar tissue formation, making it difficult to remove the tissue by a vitreous cutter and requiring the use of additional vitreoretinal instruments. As a result, the surgical procedure takes more time, and the risk of intraoperational complications is increased. Therefore, endoresection should be coupled with intraoperative procedures, inducing destruction and pathological changes in the tumour structure and then allowing easy lesion removal.ECL provides an appealing example of using this approach for cancer treatment. The approach is based on destructive chemical reactions induced by direct electric current flowing between two electrodes placed inside the tumour, resulting in tumour tissue devitalisation due to electrolysis.A growing interest in using ECL is mostly related to distinct clinical effect which, in addition to its relative affordability and availability, is shown in a great number of medical publications , 16–18.An incentivising factor that has encouraged us to develop and introduce into clinical practice the technique based on ECL application for the treatment of intraocular malignant lesions was the fact that neither such a technique nor any clinical data on its effectiveness were available.Our previous experiments involving ECL treatment of enucleated eyes with intraocular tumours demonstrated that intratumoural implantation of the electrodes (the anode and the cathode) resulted in acid-induced necrosis around the anode electrode, with subsequent formation of thickened necrotic tissue, which was difficult to remove by a vitreous cutter. Tumour tissue decomposition around the cathode was accompanied by the formation of liquid cell debris  that allowed easy lesion removal without any technical problems. This finding formed a basis for the development of an innovative combined technique that involved the use of intraocular ECL, with surface and intrastromal electrodes used during endoresection.An innovative method of positioning the electrodes during ECL allows the entire surgical procedure to be performed without any need to modify a conventional three-port endoresection technique.During the ECL procedure, liquid cell debris with totally destructed cells was formed around the intraocular cathode electrode; the resultant effect made it possible to easily remove the entire tumour mass using a vitreous cutter tip (23 G). The extrascelral position of the anode electrode, allowing the electrode relocation and sclerocompression, provided an opportunity to treat the entire scleral surface within the outline of the neoplasm projection area and, therefore, to reduce the risk of viable tumour cell survival.Intraocular water replacement with air and long-lasting silicone oil tamponade of the vitreous cavity during endoresection significantly reduced tumour cell dissemination and metastatic processes.During the ECL procedure with combined electrode positioning, we used experimentally derived electric charge parameters—20–25 coulombs. These parameters provided irreversible necrotic changes, did not lead to active bubble formation that complicated the visualisation of the ongoing processes, and were painless for the patients.To evaluate the effectiveness of the procedure, we used the method of impedancemetry for ECL exposed tissues, which allowed the dynamics of malignant tissue impedance changes to be assessed and the impedance termination moment to be determined.Z) and stable impedance readings with minimal fluctuations over time in the given portion of the tumour served as a prognostic criterion for the tumour necrosis.In the course of the ECL procedure, the intraocular electrode was moved along the area within the tumour borders, causing total lesion destruction. The resistance drop  to perform continuous-wave coagulation of the choroid around the intraocular neoplasm was also one of the ways to control haemorrhage and to facilitate the tumour endoresection, following the ECL procedure, thus localising the tissue being incised.Therefore, the modification, refinement and customising of the intraocular ECL procedure as a new eye cancer management modality help to achieve total necrosis and structural changes of intraocular lesions during the surgery and thus increase endoresection efficiency; while the development of objective and real-time assessment tools to determine the extent of pathologic changes within the tumour structures— measuring active and reactive resistivity of malignant tissue (bioimpedancemetry)—provides the basis for necessary control and monitoring during the ECL procedure.The treatment of large CM localized in the posterior pole of the eye has been and remains controversial. In our opinion, the introduction of innovative techniques into vitreoretinal surgery and the development of intraocular procedures designed to destroy tumour tissue make endoresection a promising and encouraging organ-saving treatment option.


As the proton energies currently achieved by laser driven acceleration are too low to penetrate standard tumour models on mouse legs, the aim of the present work was to establish a tumour model allowing for the penetration of low energy protons (~ 20 MeV) to further verify their effects in vivo.The long-term aim of developing a laser based acceleration of protons and ions towards clinical application requires not only substantial technological progress, but also the radiobiological characterization of the resulting ultra-short pulsed particle beams. Recent KHT mouse sarcoma cells were injected subcutaneously in the right ear of NMRI (nu/nu) mice and the growing tumours were characterized with respect to growth parameters, histology and radiation response. In parallel, the laser system JETI was prepared for animal experimentation, i.e. a new irradiation setup was implemented and the laser parameters were carefully adjusted. Finally, a proof-of-principle experiment with laser accelerated electrons was performed to validate the tumour model under realistic conditions, i.e. altered environment and horizontal beam delivery.3. The first irradiation experiment using laser accelerated electrons versus 200 kV X-rays was successfully performed and tumour growth delay was evaluated. Comparable tumour growth delay was found between X-ray and laser accelerated electron irradiation. Moreover, experimental influences, like anaesthesia and positioning at JETI, were found to be negligible.KHT sarcoma on mice ears showed a high take rate and continuous tumour growth after reaching a volume of ~ 5 mmin vitro to in vivo experimentation was for the first time realized allowing a broader preclinical validation of radiobiological characteristics and efficacy of laser driven particle accelerators in the future.A small animal tumour model suitable for the irradiation with low energy particles was established and validated at a laser based particle accelerator. Thus, the translation from  Along with the evaluation of dose delivery  and quality assurance, the biological effects on cell survival and tumour growth of laser driven particle beams have to be investigated for the whole translational research chain from basic cell experiments to animal studies and finally clinical trials[Due to their physical properties, like the inverse dose profile in tissue with an energy dependent dose deposition maximum (Bragg peak) at the end of their range, radiotherapy with protons and light ions is widely accepted to have a high potential for improving cancer treatment. However, the present accelerator technique for protons and ions demands huge buildings and high investment costs that prevent their widespread clinical application. As one potential alternative to the classical electromagnetic accelerators, particle acceleration by means of high intensity laser is under investigation,2. This l trials.in vitro studies on the radiobiological effectiveness of laser accelerated electrons[in vivo, the findings would allow transferring the existing database on RBE  values of conventional accelerated particle beams to their laser driven equivalents.Several groups have performed lectrons and protlectrons-10. Summlectrons-10. Similectrons,12. If tin vivo data have been published on laser accelerated particles. A major limitation for the in vivo application is the delivery of particle energies high enough to fully penetrate standard experimental tumours. Particularly, for laser accelerated protons, the available maximum energies of ~ 20 MeV are still too low for the homogeneous irradiation of a tumour transplanted into experimental animals. In consequence, the aim of the present study is the establishment of a tumour model that is small enough to be fully penetrated by ~ 20 MeV protons. For this, KHT mouse sarcoma cells were injected subcutaneously in mouse ears and the growing tumours were characterized with respect to growth parameters and histology; their radiation response was determined after 200 kV X-ray irradiation.So far no Finally, the applicability of the tumour model for systematic experiments at a laser accelerator was validated under the realistic and challenging conditions, like horizontal beam delivery and altered environment, at the experimental laser system JETI . Altad libitum. In order to further suppress their immune response, the mice were whole-body irradiated (WBI) with 4 Gy  two to four days before cell injection.All experiments were performed using 7-to-14-week-old female and male NMRI (nu/nu) mice continuously purchased from the Experimental Centre of the Medical Faculty of the Technische Universität Dresden. The animal facilities and the experiments at both experimental places in Dresden and Jena were approved according to the corresponding institutional guidelines and the German animal welfare regulations. The animal housing provided a 12 h light - 12 h dark cycle, a constant temperature of 26°C and a relative humidity of 50–60%. The mice were fed with commercial laboratory animal diet and water The KHT mouse sarcoma, originally described by Kallman et al., was kin-1 body weight, dissolved at 10 mg ml-1 in 0.9% NaCl; Natural Pharmacia Int., Research Triangle Park, NC, USA) and the perfusion marker Hoechst 33342  were injected one hour and one minute before tumour excision, respectively. For staining, 10 μm thick frozen sections of the tumours were cut, air dried and fixed in acetone (4°C). Finally, antibodies against pimonidazole  and against CD31 as marker for the vascular endothelium  were applied.Moreover, some of the animals were sacrificed at this time point and their tumours were extracted and immediately frozen in liquid nitrogen. The histology of the tumours was characterized by standard haematoxylin and eosin (H&E) staining. Besides, the tumour microenvironment was analysed using different immunofluorescence markers to visualize hypoxia, perfusion and vascular endothelium. The hypoxia marker pimonidazole  and 100 mg kg-1 Ketamin . The anaesthetised animals were positioned dorsoventral in specifically constructed mouse setup boxes  that was readout by an Unidos electrometer . As result, a dose rate of about 1 Gy min-1 was obtained for WBI of mice. Dose distributions and dose homogeneities within the irradiation fields were controlled with GafChromic EBT-1 films .For the present experiments, a 200 kV X-ray tube of type Maxishot 200 Y.TU/320-D03  was used for WBI and for the determination of appropriate doses for the treatment of KHT sarcoma (in Dresden). The X-ray tube was operated at 20 mA and filtered inherently with 3 mm Be + 3 mm Al as well as with the additional 0.5 mm Cu external filter. Absolute dosimetry was performed with a farmer ionization chamber , 3.8 Gy (8), 7.6 Gy (10), 9.5 Gy (8), and 13.2 Gy (7)) and one group for tumour growth control ) in order to find a suitable dose range for subsequent irradiation studies.In addition to the filters, a collimator made up of 6 mm Pb and of 2 mm Al was introduced. Four collimator openings of 6 mm in diameter allow for the irradiation of ear tumours of up to four animals protecting their bodies against radiation. The anaesthetised mice were positioned in setup boxes Figure a and werThe electron pulses were generated in a vacuum target chamber using the 30 TW laser system JETI,14 that Based on previous experiments,18,19 a For the experiments at JETI, the animals were transported from Dresden to Jena two days after injection and housed in the animal laboratory there. As a consequence of the preceding 200 kV X-ray experiments, the animals were randomly allocated in the group for tumour growth control ) and in two dose groups (0 Gy (2) and 14 Gy (9)), which were applied at the JETI laser accelerator as well as at the local 200 kV X-ray tube (see below), respectively. For treatment at JETI, the anaesthetised mice were positioned in the corresponding mouse setup boxes  was applied using the same tube parameters  and an identical setup, including mouse setup boxes and additional collimator for animal irradiation, to deliver doses of 0 Gy (2 mice) and 14 Gy (3) to the mice.After the experiment, all animals inclusive those not included in the experiment were brought back to Dresden, where the follow up took place.π/6 x a x b2, where a is the longest and b the shorter tumour axis perpendicular to a. The animals were sacrificed when the tumour reached the diameter (i.e. a) of about 10 mm or when the animal seemed to suffer. In order to assess the tumour growth delay between non-irradiated and irradiated animals, the time spans required to achieve the same relative volume increase after inclusion in the experiment were compared. Additionally, tumour volume doubling times (VDT) were estimated in the tumour volume range between 5 and 200 mm3.Tumour diameters were measured three times a week and the corresponding tumour volumes were calculated by the formula of an ellipsoid p < 0.05 were considered as statistically significant.For the graphical representation of tumour growth curves and the analysis of growth delay the software Microsoft Office Excel 2003  was applied. Statistical analysis was performed using Student’s t-test and the Welch correction implemented in the software GraphPad Prism . Values of 3  on a daily basis before starting mouse irradiation a maximum variation of the actual delivered dose of 5% was achieved.At JETI the prescribed dose of 14 Gy was delivered with a mean dose rate of 1 Gy minComparing the growth curves of the unirradiated control and the sham-irradiated "0 Gy" dose groups obtained for the two radiation sources, no apparent difference was observed Figure . In consin vitro experiments with laser accelerated protons[The integration of laser based proton accelerators in radiotherapy clinics is a long-term aim that includes not only the technological development of high intensity lasers and new techniques for dose delivery, but also comprehensive research on the radiobiological consequences of the new beam quality, i.e. ultra-short, ultra-intense particle pulses. Considering the translational research chain forgoing protons-10 have The present paper describes the establishment of a small animal tumour model that allows for the penetration by ~ 20 MeV protons. For this, an anterior mouse ear tumour model was succ3) and the corresponding tumour diameter of approx. 2 mm that allows for the penetration by ~ 20 MeV protons. By contrast, considerably higher tumour diameters of about 7 mm and tumour volumes of ~ 180 mm3 are routinely used for the standard model[3, immunofluorescence analysis revealed that already the small tumours used in the present experiments interact with the surrounding tissue and activate endothelial cells to form vessels. And, similar to larger tumours, the KHT sarcomas also generate hypoxia, which might explain their relatively high radioresistance with a tumour control dose higher than 14 Gy.Considering the established tumour model in comparison to the standard subcutaneous tumour model on mouse legs, one obvious difference is the necessity of a small tumour volume at the time of treatment ; WBI: Whole body irradiation.The authors declare that they have no competing interests.KB – participated in the design and performed all of the animal studies, performed data analysis and helped to draft the manuscript; EB – drafted the manuscript and participated in the animal studies; MB – contributed with regard to study design and scientific context; MK – contributed with regard to study design and scientific context; MO – participated in the animal studies and data analysis, JP – contributed with regard to study design and scientific context, helped to draft the manuscript; All authors read and approved the final manuscript.


Since the discovery of tumour initiating cells (TICs) in solid tumours, studies focussing on their role in cancer initiation and progression have abounded. The biological interrogation of these cells continues to yield volumes of information on their pro-tumourigenic behaviour, but actionable generalised conclusions have been scarce. Further, new information suggesting a dependence of tumour composition and growth on the microenvironment has yet to be studied theoretically. To address this point, we created a hybrid, discrete/continuous computational cellular automaton model of a generalised stem-cell driven tissue with a simple microenvironment. Using the model we explored the phenotypic traits inherent to the tumour initiating cells and the effect of the microenvironment on tissue growth. We identify the regions in phenotype parameter space where TICs are able to cause a disruption in homeostasis, leading to tissue overgrowth and tumour maintenance. As our parameters and model are non-specific, they could apply to any tissue TIC and do not assume specific genetic mutations. Targeting these phenotypic traits could represent a generalizable therapeutic strategy across cancer types. Further, we find that the microenvironmental variable does not strongly affect the outcomes, suggesting a need for direct feedback from the microenvironment onto stem-cell behaviour in future modelling endeavours. In this paper, we present a mathematical/computational model of a tumour growing according to the canonical cancer stem-cell hypothesis with a simplified microenvironment. We explore the parameters of this model and find good agreement between our model and other theoretical models in terms of the intrinsic cellular parameters, which are difficult to study biologically. We find, however, disagreement between novel biological data and our model in terms of the microenvironmental changes. We conclude that future theoretical models of stem-cell driven tumours must include specific feedback from the microenvironment onto the individual cellular behavior. Further, we identify several cell intrinsic parameters which govern loss of homeostasis into a state of uncontrolled growth. Heterogeneity among cancer cells within the same patient contributes to tumour growth and evolution. A subpopulation of tumour cells, called Tumour Initiating cells (TICs), or cancer stem cells, has recently been shown to be highly tumourigenic in xenograft models and have some properties of normal stem cells. Evidence continues to emerge that TICs can drive tumour growth and recurrence in many cancers, including, but not limited to, brain In a simplified view of the tumour cell hierarchy, TICs can divide symmetrically or asymmetrically to produce two TIC daughters or a TIC daughter and a more differentiated progeny in silico. Most germane to the loss of homeostasis is the work by Enderling et al. in vivo lineage tracing performed to date is an inability to determine the impact of microenvironmental heterogeneity on TIC symmetric division.In the HM, there are a number of cellular behaviours that govern the system. In this study, we choose to study three: the rate of symmetric versus asymmetric division of the stem cells , and TICs that are capable of forming fully invasive tumours eriments .A systematic parameter exploration of the three key parameters relating to vascularisation of the domain, symmetric vs. asymmetric division  that encode vastly different behaviour in the tissue (or tumour) arising from the TIC in question. These parameters represent different TIC phenotypes, and therefore do not represent any specific genetic mutation. In this way, we hope to generalise the intrinsic alterations which a TIC could undergo much in the same way that the hallmarks of cancer have generalised non TIC-specific alterations in vivo, the biological data to support these conclusions remains sparse. There have been some carefully undertaken in vitro experiments on single TICs in glioblastoma, a highly invasive and malignant brain tumour, which suggest that TIC specific division behaviour genetic alterations or microenvironmental perturbations. By focussing on mechanisms important for the HM of stem-cell driven tumour growth, we are seeking to identify common phenotypes which could be targeted in a variety of solid tumours in which TICs promote tumour maintenance - thereby reducing the number of therapeutic targets to a more tractable set. Only with this sort of distillation of the biological complexity inherent to cancer initiation (and indeed progression) can we hope to make progress against this disease.Our model is based on a hybrid, discrete-continuous cellular automaton model (HCA) of a hierarchically structured tissue. HCA models have been used to study cancer progression and evolutionary dynamics since they can integrate biological parameters and produce predictions affecting different spatial and time scales The continuous portion of this model is made of up the distribution and consumption of nutrients (in this case modelled only as oxygen). Vessels, which are modelled as point sources and take up one lattice point V in , are plaAny simulation performed by this model can be characterised by the parameters found in Symmetric/asymmetric division rate of stem cells Click here for additional data file.


The authors are grateful for a grant provided to CHC from Consejo Nacional de Ciencia y Tecnología (Scholarship No. 95802). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was an error in the funding statement. The correct funding statement is: The research was supported by the National Science Foundation (Grant no. 0347960 to LRG). MGS was funded in part by the European Community's Seventh Framework Programme (FP7/2007-2013;