Sodium hypochlorite is an effective irrigant for chemical debridement of root canals. However, increasing the intracanal pressure during irrigant delivery may result in irrigant extrusion into the bone and soft tissues surrounding the tooth. Because clinicians often encounter teeth with intracanal communications, the objective of the present study was to examine the effects of canal anastomosis on the generation of periapical fluid pressure at different fluid flow rates and insertion depths. Two similar polycarbonate models were used to simulate a single root with double canals, one containing, and the other without communicating channels between the canals. For both models, periapical pressure increased with increasing irrigant flow rates and insertion depths of a 30-gauge side-venting needle. In the presence of communicating channels, the magnitude of pressure build-up decreased by almost 90% irrespective of the fluid flow rate or needle insertion depth. Pressure reduction in anastomoses-containing roots provides an explanation why pressure generation in single roots is considerably higher. Nevertheless, it is still possible in teeth with canal anastomoses for pressure exceeding the intraosseous pressure to be generated when the fluid flow rate is sufficiently high and when the needle tip is close to the apical terminus. Irrigants are often introduced into root canals by positive pressure via a syringe attached to a hypodermic needle2. Sodium hypochlorite (NaOCl) is an effective irrigant for chemical debridement of root canals due to its ability to dissolve the organic components of necrotic and vital tissues, lubricate files during instrumentation, clear dentinal debris, and inactivate/dissolve bacterial biofilms3. The efficacy of NaOCl delivery is a function of the needle design, depth of irrigation needle insertion, and the flow rate of irrigant expressed from the needle4. The most difficult segment of a canal to be thoroughly irrigated is the apical third of the canal space. Even before endodontics became a dental specialty, anatomical studies have demonstrated that the greatest degree of complexity occurs in the apical third of the canal space6.Success in root canal treatment is dependent upon the clinicians’ ability to eradicate the source of intraradicular infection. Chemical debridement efficacy is related to the ability of the irrigant to infiltrate the entire canal space11, to the point where the pressure is high enough to cause the irrigant to be extruded into the periapical tissues. Zhu et al. proposed a mechanism of NaOCl accident by intravenous infusion of the irrigant beyond the root apex into the sinusoids of the medullary bone12. This mechanism, analogous to the infusion of drugs via the bone marrow spaces13, occurs when the apical fluid pressure in a root canal exceeds 30 mm Hg . When there is a patent apical foramen, NaOCl may extrude beyond the confines of the tooth and in the presence of atypical venous drainage of a tooth to the facial venous system, a full-blown NaOCl accident may occur with rapid onset of facial oedema, pain and ecchymosis. Due to the difference between intraosseous blood pressure and the central venous pressure , the NaOCl that enters bone sinusoids rapidly drains to the superficial facial venous vasculature where ecchymosis is manifested.There is a subtle balance between efficacy and safety for needle-assisted irrigation of the apical canal space. It is important to implement fluid exchange at the canal terminus by bringing the needle tip close to the working length of an instrumented canal, and by delivering the irrigant with a flow rate that is high enough to ensure bacteria and debris removal. However, the fluid flow rate of an irrigant, that of NaOCl in particular, must be controlled so that the irrigant flow is confined to, and not beyond the root canal system. There is a progressive increase in positive apical fluid pressure as irrigant flow rate increases inside the canal space2. Clinicians may be under the pretence that NaOCl accidents will not occur when a closed-end side-vented needle is not bound or locked in the canal. However, Khan et al. evaluated the fluid pressures generated in a single-canal resin model with various non-binding irrigation needles at various irrigant flow rates14. The authors opined that all positive pressure delivery systems tested were capable of generating enough apical pressure to exceed human intraosseous blood pressure (4 kPa) when the fluid flow rates were higher than 3–4 mL/min. In the single-canal polycarbonate block model used by those authors, the positive apical pressures generated by the use of a 30-gauge closed-end side-venting needle at 1 mm from the working length were much higher than those reported by Park et al. using the same needle type in a natural mandibular tooth root with two instrumented mesial canals11. In the work by Park et al., the highest positive periapical pressure obtained with insertion of a 30-gauge closed-end side vented needle to 1 mm from the working length was below 4 kPa even when high flow rates of 8 mL/min were used. Moreover, the positive periapical pressure generated with a fluid flow rate of 8 mL/min dropped to below 2 kPa when the needle was placed at 3 mm above the working length.To address both fluid exchange efficacy and safety concerns, closed-end side-venting type needles have been recommended for canal debridement, by inserting the hypodermal needle in a non-binding manner to 1 mm short of the working lengthet al. and Park et al. contained canals that were prepared in non-compliant materials. That is, the materials (polycarbonate or root dentin) could not expand to accommodate the increase in fluid volume within the canal space. Although it was not readily apparent what accounted for the discrepancy in the magnitude of positive apical fluid pressure generated from those two studies, it is speculated that the two mesial canals prepared in the natural tooth utilised by Park et al. could have been connected by canal anastomosis. This could have resulted in the displacement of fluid from one canal to the other, thereby reducing the apical fluid pressure. Because clinicians often encounter teeth that contain single roots with multiple canals, which may contain an isthmus or other types of intracanal communication, the objective of the present study was to examine the effects of canal anastomosis on the generation of periapical fluid pressure at different fluid flow rates and needle insertion depths, using a polycarbonate model to simulate a single root with double canals. The null hypothesis tested was that canal anastomosis has no effect on the magnitude of periapical pressure build-up when a root canal irrigant is delivered at different fluid flow rates and needle insertion depths.Both the models employed by Khan 16. To facilitate the development of clinically-relevant models that best approximate adjacent root canals with and without anastomoses, maxillary premolars with single roots were scanned with micro-computed tomography followed by 3-D reconstructions. One hundred and fifty-three out of the 200 randomly selected premolars were single-rooted premolars. Double canals could be identified in 84 of the single-rooted premolars, and 40.48% of those contained lateral anastomoses. Specifically, 17 of the 34 single-rooted double-canal first premolars (50%) and 17 of the 50 single-rooted double-canal second premolars (34%) had anastomosing connections in the form of discrete lateral channels and/or sheet-like isthmi. Representative examples are shown in Fig. 25.Root canal anatomy in human teeth is highly complex, as demonstrated by previous dye and micro-computed tomography studiesBased on the information derived from the 3-D rendered premolars, two models were created from polycarbonate blocks to simulate single roots with unconnected or connected double canals Fig. . The sep11. With the use of a 30-gauge side-venting needle, flow rates of 1.5 mL/min (0.025 mL/sec) and 15.6 mL/min (0.26 mL/sec) were used to represent the low-end26 and high-end11 of clinically relevant irrigant delivery rates, respectively. To achieve a flow rate higher than 15 mL/min, the 5 mL syringe described in the Experimental Setup required modification. Understandably, clinically relevant fluid flow rates may vary between these two opposing ends of the spectrum. A pilot study was thus performed, using flow rates from 1–16 mL/min and with incremental increase of 1.0 mL/min (0.17 mL/sec), to determine the sample size and to identify the lowest fluid flow rate for 4 kPa fluid pressure to be generated in each canal model. The rationale for the use of the 4 kPa cut-off was that the pressure represents the average intraosseous pressure of the sinusoids in cancellous bone27. Based on the results derived from the pilot study, a fluid flow rate of 2.5 mL/min (0.042 mL/sec) was identified as the flow rate beyond which periapical pressures exceeding 4 kPa could be generated when the side-vented needle was inserted to 1 mm above the apical terminus in the separate canal model  was significantly higher (p < 0.05) than the 8th group (3/15.6), which, in turn was significantly higher than the 3rd group (1/9.5). Periapical pressures derived from these three groups were all significantly higher than the other five groups. There was no significant difference in the maximum pressure generated by the 7th group (3/9.5) and the 2nd group (1/2.5), and between the pressures generated by the 2nd group and the 1st group (1/1.5). Pressures generated by the 1st group, 5th group (3/1.5) and the 6th group (3/2.5) were not different from one another, and were significantly lower than the pressures generated by the other 5 groups. Likewise, analysis of the data derived from the canal anastomosis model, using repeated-measures ANOVA on ranks, detected a statistically significant difference among the 8 groups (p < 0.001). Pairwise comparisons  = 3rd group (1/9.5) > 3rd group = 8th group (3/15.6) > 2nd group (1/2.5) = 1st group (1/1/5) = 7th group (3/9.5) = 5th group (3/1.5) = 6th group (3/2.5). For inter-model comparison using individual Mann-Whitney tests, a statistically significant difference (p < 0.05) was identified for each of the 8 parameter combinations  on ranks, indicated that the differences in median values among the 8 groups (2 needle insertion depths × 4 fluid flow rates) were greater than would be expected by chance; there was a statistically significant difference among those groups (p ≤ 0.001). Pairwise comparisons using the Dunn’s method Fig.  indicateons Fig.  indicateons Fig. .32. However, the relationship between fluid pressure and fluid flow rate has not been clarified. From Supplementary Fig. 28. Although water is incompressible, the Bernoulli equation should be used with reservation in accounting for fluid flow in the present situation because of potential violation of the assumptions of an inviscid fluid and steady flow characteristics. Compared with liquid helium, water is a fairly viscous fluid because the water molecules are attracted to each other by hydrogen bonds. Because effort is required to push the attracted molecules aside, there is irreversible energy loss caused by friction along the canal wall boundary during fluid movement8. The energy loss is dependent upon the Reynolds number characteristic of the velocity and the dynamic viscosity of the fluid within the system. In computational fluid dynamics simulation of needle irrigation of root canals, the fluid flow characteristic also changes from laminar to turbulent flow with increasing fluid velocities33. Notwithstanding these limitations, what the pressure sensor detects is predominantly the dynamic pressure generated by the influxing fluid head; contributions from the static pressure and hydrostatic pressure are minimal by comparison. According to Newton’s second law, this dynamic pressure is equivalent to the kinetic energy of the moving fluid mass, and is represented by 0.5 mv2, where m is the fluid mass and v is the effective fluid velocity28. The latter is the quotient obtained by dividing fluid flow rate (Q) with the net surface area (A) available for fluid flow (i.e. v = Q/A). Since the net surface area remains the same when the needle tip is fixed at a designated insertion depth, it may de deduced that the dynamic pressure generated is related to the square of the fluid velocity (v2) and hence the square of the fluid flow rate (Q2).Previous studies reported that both increased pressure during irrigant delivery and a high irrigant flow rate may cause irrigant extrusion through the root apex into the periapical regions28. Nevertheless, the similarity of the two plots indicates that the dynamic pressure recorded at a designated insertion depth is a function of the net surface area available for fluid flow. The irrigant expressed ahead of the side-vent must flow coronally and bypass the partially-occluded space created by the irrigation needle. The needle decreases the cross-sectional area of the canal, creating a bottle-neck that increases the back pressure in the region apical to the irrigation needle. The continuity equation for fluid dynamics states that mass is conserved when a fluid is in motion28. That is, under the assumption of steady flow, the product of the surface area (A) available for fluid flow and the fluid velocity (v) remains constant (A1v1 = A2v2). Thus, for a constant fluid flow rate, increase in net surface area is associated with a corresponding decrease in fluid velocity. Accordingly, the further the needle is away from the apical terminus, the lower will be the periapical pressure, due to reduction in kinetic energy of the fluid mass (0.5 mv2).Apart from fluid flow rate, dynamic pressures detected by the pressure sensor also vary with needle insertion depth. Using the separated canal model and a fixed fluid flow rate of 10 mL/min in another pilot study, the dynamic pressures generated by inserting the needle to 1–10 mm coronal to the apical terminus were recorded in 0.5 mm increments, while keeping the side-vent outlet in the same orientation. The relationship between the recorded pressures and needle insertion depth is also non-linear  may be produced even when the needle tip is more than 1 mm away from the apical terminus. Such a scenario may occur during debridement of a highly calcified canal, wherein the final shape of the instrumented canal closely approximates the geometry of the rotary instruments used for canal preparation. By contrast, chemical debridement of a single oval-shaped, non-calcified root canal is less taxing. From a pressure generation perspective, increase in the net surface area contributed by canal fins and cul-de-sacs is analogous to moving the needle further away from the apical terminus. Another “invisible” element that should be brought to attention is that, unlike polycarbonate, dentinal tubules are present in the intraradicular dentine. Patent dentinal tubules contribute to an “invisible” increase in surface area for extra pressure reduction. However, even when non-sclerotic dentinal tubules are present in the apical third of the canal space, they are packed with smear plugs immediately after mechanical instrumentation. These dentinal tubules are only rendered patent following irrigant-dependent dissolution of the inorganic and organic components of the smear layer. Hence, in theory, a root canal is most vulnerable for intracanal pressure build-up immediately following canal instrumentation. This point will be elaborated further in the discussion of the results derived from the canal anastomosis model.The relationship between net surface area available for fluid flow and periapical pressure has important clinical implications. It has been previously reported that for a 30-gauge side-venting needle, the advancing front of an irrigant expressed from the outlet of the side-vent is limited to 1–1.5 mm, beyond which efficacious debridement cannot be achievedIn the presence of communicating channels in the canal anastomosis model, the magnitude of periapical pressure build-up decreased by almost 90% irrespective of the fluid flow rate or needle insertion depth. Thus, the null hypothesis tested in the present study has to be rejected. Reduction in periapical pressures in anastomoses-containing roots provides an explanation why pressure generation in single canals is considerably higher. Although the likelihood of raising the intracanal fluid pressure to beyond the intraosseous pressure is considerably more remote in canals connected by isthmi and/or lateral channels, one can see from Fig. 34.The presence of communicating channels in the canal anastomosis model is analogous to the installation of pressure relief valves in a hydraulic circuit. These channels provide the path of least resistance for the incoming fluid to be diverted from the canal being irrigated to the second canal. Because there is less fluid moving toward the apical region, the pressure sensor detects less kinetic energy from the reduced mass of moving fluid. The extent of fluid diversion is likely to be influenced by the number of channels, the size of the channels and “loss of the channels”. In general, loss of a channel may be due to “frictional loss”, which is Reynolds number dependent, or “form loss”, which is geometry dependent, both of which have to be determined empirically. Although dentine is rigid and cannot expand or contract to alter the anatomical dimension of the bypass channels, a clinically relevant example of “frictional and form loss” may be caused by encroachment of the lateral channels by pulpal tissues, debris from mechanical preparation, or retained temporary dressings such as calcium hydroxide. These materials can decrease the functional dimensions of the channels and increase friction during fluid movement, thereby reducing the efficacy of fluid diversion. A similar reduction in anatomic diameter occurs when dentinal tubules contain intratubular materials such as collagen; decrease in functional diameter of the dentinal tubules causes substantial reduction in the hydraulic conductance of dentineIn the present study, the side-vent outlet was oriented 180° from the bypass channels during all pressure measurements (see Methods). Paradoxically, we found that even with the use of a fixed flow rate and a fixed needle insertion depth, the recorded pressures may vary substantially with the orientation of the side-vent outlet. This is illustrated by the results of a preliminary study comparing the periapical pressures recorded in the canal anastomosis model, with the side-vent facing the communicating channels or oriented 180° from the channels  for micro-computed tomography. The premolars were scanned  with a nominal isotropic resolution of 15 μm inside a 48-mm diameter scanning vial at 90 kVp, 88 mA, 8 W, 500 msec integration time and 500 projections per 180°. One hundred and fifty-three premolars with singled-rooted roots were detected, of which 84 teeth had double canals. Three-dimensional representations of each root canal system was rendered with VGStudio MAX 2.1 .The models were each constructed from two separate blocks of impact-resistant polycarbonate . For the separate canal model Fig. , left, eThe final separate canal model consisted of two separate simulated instrumented root canals: 1) the test canal on the left Fig. , right. 31; clinically, these rates are only manually possible by using a 1 mL syringe. To ensure delivery of the exact volume of irrigant, no air bubbles were allowed to enter any part of the delivery system. This criterion was fulfilled by using a 50 mL syringe , needle insertion depth  and irrigant flow rate  on the maximum periapical pressure generated in the test canals. However, the normality and equal variance assumptions of the data sets were violated even after nonlinear transformations. Hence, data sets from the separate canal model (2 needle insertion depths × 4 fluid flow rates) were analysed with repeated measures ANOVA on ranks and Dunn’s multiple comparison tests, taking into consideration that measurements were repeatedly recorded within the same experimental setup. Likewise, data sets from the separate canal model (2 needle insertion depths × 4 flow rates) were analysed with repeated measures ANOVA on ranks and Dunn’s multiple comparison tests. For comparison between the separate canal model and the canal anastomosis model, the two data sets generated using a particular needle insertion depth and fluid flow rate were analyzed with the Mann-Whitney rank sum test. For all analyses, statistical significance was pre-set at α = 0.05.A three-factor analysis of variance (ANOVA) was initially planned to examine the effects of the ability for collateral redirection of an incompressible fluid (separate canal model Supplementary InformationSupplementary Video S-1aSupplementary Video S-1bSupplementary Video S-1cSupplementary Video S-1d


The following grant numbers from the funder Chang Gung Memorial Hospital are missing from the Funding section: CMRPG6C0061, CMRPG6C0062, and CMRPG6C0063


By staining thin (5–10 µm) paraffin and thick (50 µm) agarose tissue sections, we detected HER2- and CEA-positive human tumour cells infiltrating the surrounding tissues or metastasizing to different organs, including the brain, testis, lung, liver, and lymph nodes. Compared to conventional fluorescently labelled antibodies the sdAb-HER2-QD and sdAb-CEA-QD nanoprobes are superior in detecting micrometastases in tissue sections by lower photobleaching and higher brightness of fluorescence signals ensuring much better discrimination of positive signals versus background. Very high two-photon absorption cross-sections of QDs and small size of the nanoprobes ensure efficient imaging of thick tissue sections unattainable with conventional fluorescent probes. The nanobody–QD probes will help to improve early cancer diagnosis and prognosis of progression by assessing metastasis.Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies  and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in  To improve cancer prognosis and, hence, patients’ survival, early detection of the disease is one of the main purposes in diagnostic approaches. In this regard, the rapidly progressing field of nanotechnology is considered a powerful tool in cancer diagnostic and therapeutic applications3. The development of new nanoprobes which can detect cancer biomarkers with high specificity and sensitivity is appealing. The use of nanomaterials brings a number of advantages not only in diagnostics, but also in therapeutic concepts, such as improvement of water compatibility of hydrophobic drugs, their protection from degradation, with the result of a longer release, increase in drug bioavailability, tumour selectivity, and greater penetration depths for the treatment of deep seated tumours5. Among the enormous variety of nanoprobes available, semiconductor quantum dots (QDs) exhibit advanced optical properties and attract increasing attention for applications in biomedical research, such as cancer marker detection and tumour cell imaging6. QDs are a class of highly fluorescent homogeneous semiconductor nanocrystals 2–10 nm in size, composed of a photoluminescent semiconductor core coated with a shell of another semiconductor with a larger bandgap7. QDs have been shown to have broad absorption spectra, which allow excitation of QD of different sizes (fluorescence emission) at the same wavelength remote from their respective narrow emission bands, resulting in emissions of different fluorescence colors that can be detected simultaneously. Further advantages are a quantum yield close to 100%; high single-photon and two-photon molar extinction coefficients (orders of magnitude higher compared to organic dyes) and a high resistance to photobleaching and chemical degradation9.Cancer still remains a great challenge to public health and represents a giant economic burden to the society. In 2013, more than one and a quarter million people died from cancer in the European Union10. sdAbs are characterized by a low tendency to aggregate; they diffuse much better into tissues than full-size IgGs, and their size allows the labelling of thicker tissue segments than conventional IgGs do11. Because of these advantages, we have recently conjugated QDs to nanobodies in a highly oriented fashion, with all antigen binding sites facing outwards, which considerably increases the nanoprobe sensitivity for diagnostic and possible therapeutic use in oncology13 and demonstrated their advantages in breast and lung cancer cell imaging14.To ensure cell specificity, QDs are normally bound to recognition molecules, such as antibodies, aptamers or peptides. Here, we used antibody fragments derived from llama antibodies, often called “nanobodies”, which are much smaller than conventionally used full-size IgG antibodies, with a molecular weight of only 13 kDa. These single-domain antibodies (sdAbs) are the smallest antibody fragments capable of binding their antigens with affinities comparable to conventional antibodies16. Another glycoprotein most widely used as a tumour marker is CEA, whose level has been reported to be elevated in colorectal, breast, lung, pancreatic, liver, stomach, thyroid, prostate, bladder, and ovarian cancers17. CEA is often expressed in pancreatic ductal adenocarcinoma (PDAC), which accounts for over 90% of all pancreatic cancers, a devastating malignancy with extremely poor prognosis. PDAC normally grows fast, metastasizes early, and is generally accompanied by noticeable resistance to adjuvant therapeutic strategies18. In clinical oncology, CEA is commonly used as a serum marker as part of both preoperative staging and follow-up of the response of cancer patients to surgery and chemotherapy19.Among the large number of tumour-associated antigens that can be targeted, HER2 receptor was found to be overexpressed on the cell membrane of various tumour types, including lung, ovarian, stomach, uterine, and especially breast cancers, where 25–30% of cells are HER2-positive. The expression level of this protein in breast cancer patients is mostly used to select patients for treatment with trastuzumab (Herceptin), a monoclonal antibody that recognizes and blocks the HER2 protein expressed on tumour cells23 and MDA-MB-231 cells, which express low levels of HER224. In the BT474 model, implantation of human breast tumour cells together with estradiol/norgestrel hormone pellets results in an increase in its metastatic potential25. Like these breast tumour models, PDAC mouse models are very invasive: AsPC1 and Capan-1 cells express high levels of CEA receptor, and, particularly, the AsPC1 model starts to develop symptoms as early as 4 to 5 weeks after implantation, with massive invasion in the neighbouring organs26. In this study we have shown that QDs conjugated in a highly oriented manner with the sdAbs targeting either HER2 or CEA are powerful tools for detecting human micrometastases and disseminated HER2- or CEA-positive tumour cells even in thick tissue sections of biological samples of metastatic breast and pancreatic tumour mouse models, respectively.Different metastatic mouse models of breast tumours have been described, such as the transplantation of BT474 and SKBR3 cell lines, which express high levels of HER2 receptorApproximately 4–5 weeks after implantation of BT474 human breast tumour cells, primary tumours developed in the right inguinal mammary gland, accompanied by an enlargement of the inguinal lymph nodes Fig. . RepreseWe determined the capability of sdAb-HER2-QD nanoprobes to bind to HER2 receptors expressed on tumour cells. The cross reactivity of the antibodies was tested and proved to not react against mouse tissue (Data not shown). Thin agarose sections (20 µm) of tumours expressing high and low HER2 levels, BT474 and MDA-MB-231, show HER2 in green fluorescence, stained with the anti-HER2 polyclonal antibody NeuC-18 (pAb-HER2) in combination either with a secondary antibody labelled by Alexa Fluor (AF)−546 (pAb-HER2-AF) or with sdAb-HER2-QDs nanoprobes . When negative controls, i.e. unconjugated QDs (UN-QDs) or QD conjugated to irrelevant sdAbs (gp120-QDs) were applied onto thin agarose sections of high-HER2-expressing tumours, no comparable signals at the tumour cell membrane were detected , which reflected the high specificity and sensitivity of sdAb-HER2-QDs for binding to HER2-expressing tumour cells. Although we detected autofluorescence on BT474 tumour cells , this signal is negligible in comparison to the signal generated by the binding of the sdAb-HER2-QDs . The staining of thin agarose sections of MDA-MB-231 tumours, which express low levels of HER2, with sdAb-HER2-QD nanoprobes did not result in any membrane stain either .Confocal microscopy images Fig.  using eiblue curve) a fluorescence emission peak was detected at 570 nm. Instead, when the samples where stained with UN-QD nanoprobes, no specific signal of the QDs was detected on the tumour sample , thereby demonstrating the specificity of sdAb-HER2-QDs binding.The sdAb-HER2-QD nanoprobes are characterized by a broad absorption spectrum and a narrow emission peak Fig. . The resIn order to evaluate the feasibility of using the sdAbs-QD conjugates in thick tissue sections of 50 µm and to explore the potential advantage of the small size of the nanoprobes for two-photon imaging of deeply seeded tumours, we embedded the tumours in agarose before immunostaining using the free-floating protocol. Figure right column).Figure To investigate whether sdAb-HER2-QDs could detect small metastases with high signal intensity and high specificity in deep tissue, we used 50-µm agarose slices of lymph nodes infiltrated by tumour cells from the BT474 and SKBR3 human breast tumour mouse models. In Fig. The capacity of sdAb-HER2-QDs to specifically detect disseminated tumour cells was not limited to lymph node metastases, but was also shown for metastases in the liver, duodenum, lung, testis, and brain in the SKBR3 xenograft tumour mouse models Fig. . ImmunohTo evaluate the capacity of sdAb-CEA-QDs to detect CEA-positive human tumour cells invading different mouse tissues, we obtained biological samples from three metastatic PDAC mouse models orthotopically transplanted. Figure first column and higher magnification picture). A small group of metastases was also detected as bright green signals by confocal microscopy in the liver of the Capan-1 tumour mouse model stained with sdAb-CEA-QDs . In control tissue sections from MIA Paca-2 tumours with low expression levels of the CEA antigen, no positive staining with sdAb-CEA-QDs was detected . The use of the negative control nanoprobes, UN-QDs and gp120-QDs, on thin paraffin sections of the high-CEA-expressing Capan-1 tumour yielded no positive signal, thereby confirming that QDs do not unspecifically bind to tumour cells .In paraffin tissue sections of the AsPC1 tumour mouse model, the invasion front consisting of tumour cells growing into the intestinal crypts was seen as intense green fluorescence signals, due to the specific binding of the sdAb-CEA-QD nanoprobes to human tumour cells . Characterized by a diameter below 12 nm, these nanoprobes are composed of a QD conjugated with four homogeneously oriented copies of sdAbs13. These highly oriented sdAb-QD nanoprobes have already been shown to have a very broad absorption band, with the highest extinction in the UV spectral region, contrasting with have the narrow absorption range of Alexa fluorophores of about 130 nm14. Although the absorption band of QDs is very broad, their emitted fluorescence can be tuned with high precision by varying the size and composition of the fluorescent core. Thus, when the core of QDs is composed of CdSe, a variation of core size in the 2 to 8 nm range gradually shifts the emission peak of such QDs from 480 to 660 nm28. This constitutes a clear advantage of the QDs over Alexa fluorophores when used in multiplexed applications, since detection of different antigens in the same sample by using QD-based nanoprobes is possible with the use of a single light source. Like other organic fluorophores, the pAb-HER2 and AF546-labelled secondary antibody combination used in this study would be poorly suitable for multicolour signalling at a single wavelength. In contrast, all sdAb-HER2-QD nanoprobes are excited at 405 nm, and the emitted fluorescence is tuneable by varying their core size , making them emitting at different wavelengths of the spectrum12.Our study shows that QD-labelled sdAbs targeting the tumour-associated antigen HER2 or CEA are highly efficient tools for the detection of 6.The 2P-LSM excitation is restricted to the focal plane, which entirely eliminates the out-of-focus signal, resulting in an increased resolution and less photobleaching. The excitation is performed using much longer wavelengths (700–1000 nm) which is known to penetrate tissue more deeply causing less light scattering and better signal-to-noise ratio. In our samples, we observed less photobleaching when using sdAb-HER2-QD nanoprobes compared with pAb-HER2-AF. We believe that this is related to the QD capability of producing stronger emission, in combination with their higher photostability due to the inorganic nature of the QD fluorophore. We have also shown that the use of sdAb-HER2-QDs is highly efficient in detecting circulating tumour cells and metastatic spread in lymph nodes of two xenograft breast tumour models. In both lymph nodes, we were able to clearly distinguish between HER2-positive tumour cells, which were specifically stained by the nanoprobes, and unstained leucocytes, which did not express the antigens. Strikingly, the 50-µm sections obtained from agarose-embedded tumours ensured a better preservation of the morphological structures of the tissue and higher quality of the fluorescent signals compared to thinner paraffin slices. SdAb-HER2-QDs have a higher diffusibility in thicker sections compared to the commercial pAb-HER2-AF, which makes them excellent probes for deep tissue imaging.Our results also show that imaging of QDs using 2P-LSM results in a much slower attenuation of the fluorescence emission intensity when moving deeper into tissue as compared with the single-photon confocal microscopy, allowing the detection of signals from sources located farther from the tissue surface. This can be explained by one of the advantages of excitation with 2P-LSM over the excitation with confocal microscopy, namely, nonlinear excitation, which generates fluorescence emission proportional to the square of illumination intensityIn the tumour samples studied, the nanoprobe fluorescence was reduced by ~50% of the initial emitted fluorescence at a depth of 10 µm when imaged by 2P-LSM. For comparison, the fluorescence emitted by pAb-HER2-AF was completely abolished at this depth. This is accounted by the difference in fluorescence decay and light scattering through the thick tissue section. The ability of the antibody to penetrate the tumour tissue section was probably impaired by the size and structure of the antibody and the complex composition a tumor presents. Many clearing methods, which improve imaging of the tissue allowing better light penetrance, exist and have often been used, however there are still not stablished protocols for clearing tumours tissues and which would not interfere with the architecture of the extracellular matrix. As we were interested in preserving such structures for later analysis of tumour cell - extracellular matrix interactions, such techniques were not desirable for our purposes.et al. (2014), the lifetime of sdAbs-QD nanoprobes is about 24 ns . This is significantly higher than that of mAb-HER2-AF, which has been found to be about 3–4 ns, similar to the lifetime of the autofluorescence of cellular components, such as nicotinamide adenine dinucleotide (NADPH), with a fluorescence lifetime of approximately 2 ns. Thus, QD-based nanoprobes can ensure better sensitivity when used in combination with imaging techniques29. This characteristic is essential for the analysis of tumour tissues, which display alterations in the concentration and constitution of extra- and intracellular components, such as collagen, elastin, keratin, haemoglobin, and NADPH, leading in some cases to substantial autofluorescence30.As previously reported by Rakovich In this study, we demonstrated that sdAb-CEA-QDs specifically detect tumour cells in tissues of metastatic models of PDAC tumours. The invasion of tumour cells into healthy organs/tissues surrounding the primary tumour is reliably detected by their labelling with the developed nanoprobes. Overall, the small size of the nanoprobes is an advantage over full-size antibodies, allowing a higher diffusion of the probes through tissues, and their retention on tumour cells within a healthy tissue.We have demonstrated that sdAb-QD conjugates are effective tools to detect disseminated human tumour cells and micrometastases by binding HER2 and CEA, two antigens overexpressed in metastatic breast and pancreatic tumours, respectively. SdAb-QDs are shown to be largely superior to commercial polyclonal antibodies conjugated to conventional Alexa dyes. The 2P-LSM technique is shown to have considerable advantages over single-photon confocal microscopy to visualize the nanoprobes on metastatic tumours, since the emitted fluorescence generated by 2P-LSM is higher, resulting in a much better signal-to-background ratio. Additionally, the diffusibility of sdAb-QD nanoprobes through tissue facilitates the access to small metastases and complex structures, making these nanoprobes powerful tools for cancer diagnostic and therapeutic applications.2, were harvested at ~80% confluency by brief trypsinization in a 0.05% trypsin–EDTA solution, washed several times, and placed into sterile PBS shortly before implantation.Human PDAC cells AsPC1 (CRL-1682), Capan-1 (HTB-79), MIA Paca-2 (CRL-1420) and the human breast cancer cells BT474 (HTB-20), SKBR-3 (HTB-30), and MDA-MB-231 (HTB-26) were purchased from ATCC . KPL-4 cells were provided by J. Kurebayashi . BT474 and AsPC1 tumour cells were cultured in RPMI 1640 medium with 10% foetal bovine serum ; MDA-MB-231 cells, in DMEM medium with 10% FBS; SKBR3 cells, in McCoy’s 5 A medium with 10% FBS; Capan-1, in IMDM medium with 20% FBS; and MIA Paca-2, in DMEM/F12 medium with 10% FBS and 2.5% horse serum (Biochrom). All media were from Life Technologies. All cells were maintained at 37 °C in a humidified atmosphere with 5% CO34.Highly fluorescent QDs emitting at 570 nm were conjugated to sdAbs targeting HER2 and CEA, or to the protein gp120 through an engineered additional C-terminal cysteine residue. The protocols for sdAb development and isolation, QD synthesis, solubilisation in water, stabilization, and conjugation with sdAbs were described previouslyad libitum. For tumour cell implantation 6–12 weeks old nude NMRI-Foxn1nu mice (Charles River Laboratories) were anesthetized by intraperitoneal (i.p.) injection of 15 mg/kg xylazine (Ecuphar) and 75 mg/kg ketamine (Medistar) as described previously35.Animals were housed in individually ventilated cages and allowed food and water 6 BT474 cells resuspended in 20 μL of PBS or 5 × 106 KPL-4 cells resuspended in 30 μL of PBS were orthotopically transplanted into the right abdominal mammary fat pad. For negative controls, 1 × 106 low-HER2-expressing MDA-MB-231 cells were implanted orthotopically to generate xenograft tumours with a low HER2 expression.For the BT474 mouse model, two hormone pellets were implanted at the neck of female athymic nude mice, each pellet containing 17β-estradiol and norgestrel . Subsequently, 1 × 10scid IL2Rγnull (NSG) mice obtained from Jackson Laboratories as described earlier . The animals were bred and kept in a specialized pathogen-free facility at the University of Regensburg. For obtaining xenograft mouse models, the animals were irradiated (1 Gy) and, 3 h later, transplanted with 3 × 106 SKBR3 tumour cells. In all experiments, cells were transplanted into the liver of new-born mice. The mice were euthanized and analysed at the age of 3–5 months after transplantation23.The second model used was the high-HER2-expressing SKBR3 xenograft model, using NOD-Foxn1nu mice (Charles River Laboratories), were orthotopically transplanted into the head of the pancreas with 1 × 106 AsPC-1 or Capan-1, high-CEA-expressing human PDAC cells or negative controls, MIA Paca-2 low-CEA-expressing, all resuspended in 20 μL of PBS as described before18.10–20 weeks old male athymic nude NMRI-2 asphyxiation and cervical dislocation mice were dissected and tumour sizes, numbers and locations of macroscopic metastases were assessed. All animal experiments were performed in accordance with German animal ethics regulations and were approved by the local ethics office of Lower Saxony .For analgesia, the mice received Rimadyl  i.p. during surgery and Metamizole  in drinking water for two days before and for three days after transplantation. Mice were examined three times a week for weight loss, general condition, and palpable tumour formation. After euthanization by COBlood was obtained by cardiac puncture from mouse with a heparin filled syringe. To obtain the cells from the ascites, several washes were performed at the abdomen cavity and blood and cells from ascites were prepared as smear slides.g. The supernatant was used as the total cell extract. The protein concentration was determined using BCA Protein Assay Reagent (Pierce).To obtain cell lysates, cultures were washed twice with PBS, incubated in 3 mL of lysis buffer (50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 5 mM EDTA, and 1% Triton X-100) containing a protease inhibitor cocktail (Roche) for 30 min and centrifuged for 15 min at 21,300 Samples of 30 µg of total protein extracts reduced by 50 mM of sample reducing agent , were separated by SDS-PAGE  and transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were blocked with 5% bovine serum albumin  in 0.05% TBS-T  for 1 h at room temperature (RT), probed for 2 h with either an anti-HER2 rabbit polyclonal antibody :sc-284, Santa Cruz Biotechnology, dilution of 1:200) or a an anti-CEA rabbit monoclonal antibody , each diluted in blocking solution. Mouse monoclonal anti-β-actin antibody was used as protein loading control . Blots were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody  and developed using chemiluminescent HRP substrate (Millipore Immobilon system). Signals were detected by using a Bio-Rad Chem-Doc luminescence detection system.Tumour tissue and organs from all xenograft models were harvested and fixed overnight (ON) in 10% formalin at 4 °C. After that, the tissues were kept at 4 °C in a PBS containing 0.1% of sodium azide for preservation. The samples were embedded either in paraffin or in 5% agarose blocks for preparation of serial sections. From all models, 5-μm paraffin tissue sections were prepared, stained with haematoxylin and eosin (H&E), and examined by routine light microscopy. For IHC, 5-μm paraffin sections were de-paraffinized in xylene, rehydrated through ethanol , and finally placed in TBS. Antigen retrieval was performed in a citrate buffer (2% citric acid and 8% sodium citrate) for 60 min at 95 °C and then 20 min at RT. The slices were incubated with 3% hydrogen peroxide inhibitor (DAKO) to quench the endogenous peroxidases and washed in TBS. Nonspecific binding was blocked by incubating the sections for 1 h in Sea block solution (ThermoScientific). For tissue IHC, either anti-Ki-67 rabbit monoclonal antibody  or anti-HER2 rabbit monoclonal antibody  was used ON at 4 °C. The slices were washed two times for 5 min in a Tris-buffer solution and incubated with secondary HRP-conjugated anti-rabbit antibody (Nichirei) for 1 h; the signals were developed with 3-amino-9-ethylcarbazole (AEC). The slides were counterstained with haematoxylin and mounted with Aqua Tex (Merck).IHC with anti-HER2- or anti-CEA-sdAb-QDs (15 nM) conjugated nanoprobes were performed for 1 h in a humidified chamber at RT. Tumour tissues as negative controls were stained with a solution of either unconjugated (UN)-QDs or QDs conjugated to gp120 protein (gp120-QDs) . For all fluorescent conjugates, the slices were washed three times in TBS before nuclear staining was performed with DRAQ5 , and sections were mounted using Cytoseal (Thermo Fisher Scientific).Agarose tissue sections (30–50 μm) were obtained with a Vibratome (Leica VT1000S) and washed three times in TBS-T. All incubations were performed free-floating in solution, first with 3% hydrogen peroxide inhibitor (DAKO) followed by a washing step with TBS-T before. Nonspecific binding was blocked by incubation for 1 h in 2% BSA in TBS with 0.5% Tween (TBS-T). For tissue labelling, the agarose tissue sections were incubated with either anti-HER2 polyclonal antibody (pAb) (Neu (C-18):sc-284, Santa Cruz Biotechnology, 1:200) or anti-HER2 monoclonal antibody  and then with Alexa Fluor 546 (Thermo Fisher Scientific) or anti-HER2-QDs (15 nM) for 2 h. Negative controls were stained with a solution of either UN-QDs or gp120-QDs (15 nM). Nuclear staining was performed with DRAQ5 , and tissue sections were mounted using Cytoseal (Thermo Fisher Scientific).First, a Leica SP5 confocal laser scanning microscope  equipped with hybrid detectors, a 40× immersion objective lens (HCX PL APO lambda blue), a motorized stage, and a tuneable laser (470–670 nm) was used. QDs, Alexa Fluor 546, and DRAQ5 were excited at 405, 561, and 647 nm, respectively, and their individual emissions were collected at 560–580, 560–620, and 665–795 nm. Second, a custom-made two-photon laser scanning microscope (2P-LSM) equipped with a femtosecond-pulsed titanium–sapphire laser  and a Zeiss W Plan Apochromat 20× (NA 1.0) water immersion objective lens was used. For excitation of the QDs, Hoechst and Alexa 546 the laser was respectively set at 850 nm and at 830 nm. The emitted light was split by two longpass dichroic mirror (Semrock) one 560 nm, and another 405 nm, and collected by photo-multiplier tubes (Hamamatsu). For the z-stacks acquisitions no laser power depth compensation was performed. All images were processed with the FIJI (an image-processing package based on ImageJ), Imaris 8.30 (Bitplane), Graph Pad Prism 6  and Matlab  software. For 3D imaging of microscopy z-stacks as volumes, the Java-based ImageJ 3D Viewer plugin developed by Benjamin Schmid (Biozentrum Universität Würzburg) was used.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary InformationSupplementary V1Supplementary V2


Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 106 mm2 s−1), vascular perfusion/permeability  and fractional blood volume  respectively. Pathological correlates were sought, and preliminary gene expression profiling performed.Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kgP<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour Ktrans (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis.Treatment with vanucizumab, bevacizumab or LC06 induced a significant (Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition. Angiogenesis, the development of new blood vessels to provide a nutritive blood supply, is a prerequisite for solid tumour growth and survival . AngiogeThe binding of vascular endothelial growth factor-A (VEGF-A) to its endothelial receptors is a key driver of tumour progression, angiogenesis and vascular permeability. Vascular endothelial growth factor is considered the most potent angiogenic growth factor, whose expression is also associated with poor prognosis in a wide range of human cancers .The contribution of the Tie-2 receptor ligands angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) to tumour vascular remodelling has also been established . Ang-1 iVanucizumab , a novel heterodimeric bivalent bispecific human antibody against Ang-2 and VEGF-A (bevacizumab-based) has recently been described  provide a means of defining noninvasive quantitative biomarkers to inform on biologically relevant structure–function relationships in tumours. This enables an understanding of the behaviour and heterogeneous distribution of such associations, and informs on response and resistance to treatment . For theex vivo histopathology. Differences in the sensitivity of functional MRI biomarkers for assessing tumour response to the dual targeting of Ang-2 and VEGF-A are revealed. Preliminary analyses of vascular-related tissue biomarkers associated with tumour response to vanucizumab, identified by gene expression profiling, are also described.Vascular endothelial growth factor inhibitors including bevacizumab have been shown to elicit a tumour antivascular response that is detectable by dynamic contrast-enhanced (DCE) MRI  and maintained at 37 °C in a humidified incubator with an atmosphere of 95% air, 5% CO2. Cells were authenticated by short tandem repeat (STR) profiling by ATCC and were not passaged for >6 months in our laboratory.Colo205 human colorectal cancer cells , were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 2 mnu/nu mice  were injected with 5 × 106 Colo205 cells subcutaneously in the right flank. Tumours were selected for imaging when volumes reached ∼250 mm3, as assessed by callipers, using the formula for ellipsoid volume, (L × W2)/2, where L and W were the two largest dimensions of the ellipsoid. Immediately following pretreatment MRI (day 0), mice were randomised and administered with a single 10 mg kg−1 intraperitoneal dose of either vanucizumab , bevacizumab , LC06 (anti-murine/human Ang-2)  Act 1986, the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research (−1) plus fluanisone ), midazolam ) and sterile water (1 : 1 : 2). A lateral tail vein was cannulated with a 27G butterfly catheter  for remote administration of MRI contrast agents. Mice were positioned in the coil on a custom-built platform to isolate the tumour, and their core temperature was maintained at 37 °C with warm air blown through the magnet bore.Magnetic resonance imaging was performed on a 7T horizontal bore Bruker microimaging system  using a 3 cm birdcage volume coil. Anaesthesia was induced with an intraperitoneal injection of fentanyl citrate  images were then acquired from three 1 mm thick axial slices across the centre of the tumour using an echo-planar imaging (EPI) sequence . Diffusion-weighted MRI exploits the random diffusion of water molecules to measure changes in tissue cellularity, quantified through the measurement of the apparent diffusion coefficient  , and 60 dynamic scans  acquired for 3 min before and 17 min after an i.v. injection of 0.1 mmol kg−1 of a clinically approved, low-molecular-weight contrast agent Gd-DTPA  (Ktrans (min−1). Increasing contrast agent concentration in the extracellular leakage space is related to both tumour perfusion and permeability; an effective vascular targeting drug would however be expected to reduce Ktrans  true-FISP sequence with one baseline scan  or omalizumab (n=7). Multigradient-recalled echo (MGRE) images  were acquired from 3 contiguous 1 mm thick axial slices through the tumour, using a 128 × 128 matrix over a 3 × 3 cm field of view. A second set of identical MGRE images were acquired 2 min after intravenous injection of 150 μmolFe kg−1 USPIO particles . The USPIOs create magnetic susceptibility variations in the proximity of perfused blood vessels, increasing the transverse MRI relaxation rate R2* of water in the surrounding tissue. Their long intravascular half-life enables steady-state measurements of tumour R2* to be made that are used to determine fractional blood volume   particles as an intravascular MRI contrast agent, was performed on a separate cohort of Colo205-bearing animals. Tumours were imaged at baseline (day 0), and 5 days post treatment with a single i.p. dose of 10 mg kg2-weighted images for each tumour-containing slice, where clear delineation of tumour from surrounding tissue could be determined. Diffusion-weighted, DCE and susceptibility contrast MRI data were fitted on a pixel-by-pixel basis using in-house software . Diffusion-weighted and MGRE images were fitted using a Bayesian maximum a posteriori approach, allowing estimates of the median ADC and USPIO-induced change in R2* (ΔR2*) to be calculated, respectively  of the pulse sequence and incorporating the Tofts and Kermode pharmacokinetic model, providing estimates of Ktrans .Tumour volumes were determined using segmentation from regions of interest (ROIs) drawn on Tectively . Fractio−1; Hypoxyprobe Inc., Burlington, VT, USA) , a marker for perfused vessels, was injected via a lateral tail vein  attached to a BX51 microscope , driven by image analysis software  as previously described  and pimonidazole adduct area (hypoxia) using CellP as previously described (α-smooth muscle actin (α-SMA) immunohistochemistry was performed on 5 μm thick formalin-fixed, paraffin-embedded (FFPE) sections using mouse monoclonal anti-α-SMA antibodies  and an anti-mouse HRP-labelled polymer . The percentage of α-SMA-positive stained area was determined by analysing ⩾5 randomly selected fields at high magnification (× 200) from each tumour section using a colour deconvolution program in ImageJ . An adjacent section was stained with H&E and imaged under bright-field illumination. The percentage of tumour necrosis was determined using CellP as previously described . Total RNA was isolated from frozen xenograft tissues following standard procedures . RNA integrity was assayed using the Agilent Bioanalyzer system , quantitated , and 500 ng of polyadenylated RNA passing the QC criterion of RIN >7 was subjected to sequencing library generation using Illumina TruSeq RNA sample preparation kit v2  according to the manufacturer's instructions without modification. Final cDNA libraries were analyzed for size distribution, quantitated by qPCR, and then normalised to 2 nM in preparation for sequencing. Libraries were clustered using Illumina TruSeq PE Cluster Kit v3. Finally, the DNA sequence was determined on the HiSeq2000 using sequencing-by-synthesis technology via the Illumina TruSeq SBS Kit. For each specimen, 2 × 50 bp paired-end sequences were yielded at an approximate coverage of 50 million paired-end reads.All RNASeq samples passed quality control in terms of number of reads per sample and read quality. Short reads were aligned to the human and mouse transcriptome (based on Ensembl v60) using Bowtie2 . Based oP-value of <5% considered significant. The ANOVA was applied on log2-transformed RPKM values for vanucizumab, bevacizumab or LC06 treated against control samples in order to identify differentially regulated genes. Multiple comparison correction was implemented across contrast pairs. Implementation of ANOVA for gene expression data was via R function aov (http://www.statmethods.net/stats/anova.html).Statistical analysis of MRI and histological data was performed with GraphPad Prism 5 . The mean of median values were used for statistical analysis of all quantitative MRI data, apart from tumour volumes, where mean values were used. A two-way ANOVA with Bonferroni correction, nonparametric Wilcoxon matched pairs signed-rank test or one-way ANOVA with Dunnett's multiple comparison test were used where appropriate, with a 2-weighted MR images before and 5 days post treatment with a single dose of vanucizumab, bevacizumab, LC06 or omalizumab. Representative T2-weighted images from all treatment groups are shown in P<0.01) cytolentic response (mean tumour volume increased by 52%) compared with control (166%). Treatment with either bevacizumab or LC06 induced a similar response, with significantly (P<0.001) lower mean tumour volumes compared with control  (Tumour volumes were determined from multislice T06: 74%) . No adveP>0.05).Diffusion-weighted MRI was used to explore whether the antitumour effects of vanucizumab resulted in changes in ADC, an imaging biomarker associated with alterations in cell density and which typically increases with successful treatment. The mean ADC for each treatment group, at baseline and 5 days post treatment, are summarised in Ktrans are shown in Ktrans and IAUGC60 is summarised in P<0.05) reduction in both Ktrans and IAUGC60 was determined in the vanucizumab-treated cohort only. There was however no significant difference in either DCE MRI biomarker 5 days post treatment with vanucizumab, bevacizumab or LC06 when compared with omalizumab (control).Dynamic contrast-enhanced MRI was used to assess any vascular response following treatment with vanucizumab, bevacizumab, LC06 or omalizumab. Representative parametric maps of P<0.01) reduction in mean tumour fBV after 5 days of treatment (P<0.05) greater in the vanucizumab cohort (−2.7±0.5%) compared with that determined in the omalizumab cohort (−0.7±0.6%).Representative parametric fBV maps acquired before and 5 days post treatment with either vanucizumab or omalizumab are shown in reatment . The redP<0.05) lower tumour perfusion relative to control (vanucizumab: 17±3% bevacizumab: 15±3% control: 28±4%). There was no significant difference in hypoxia in any of the treatment groups compared with control. A single dose of either vanucizumab or LC06 had no significant effect on MVD 5 days after treatment compared with control (vanucizumab: 15±1% LC06: 16±1% control: 18±1%). In contrast, treatment with bevacizumab resulted in significantly (P<0.05) lower mean MVD compared with control (bevacizumab: 14±1%). Analysis of pericyte coverage revealed no significant difference in the expression of α-SMA following a single dose of vanucizumab, bevacizumab or LC06 compared with control. Haematoxylin and eosin staining revealed small localised regions of tumour necrosis (<20%) in all treated and control tumours. There was no significant difference in the mean percentage necrosis at 5 days post treatment in any treatment group compared with control.Representative images acquired of each histological marker assessed for all treatment groups and control tumours are shown in P<0.05 and |log2 ratio|>0.5 , also aligning with the significantly lower tumour uptake of Hoechst 33342. The susceptibility effects generated by USPIO particles are stronger than gadolinium chelates used in DCE MRI, and thus arguably may provide a more sensitive measurement of tumour response to antivascular/antiangiogenic therapy , and shown to inform on both incipient tumour angiogenesis , as measured by Hoechst 33342 uptake, were associated with a nonsignificant increase in hypoxia in all treatment groups compared with control. Previous work has shown that vanucizumab causes a transient increase in expression of the hypoxia-regulated protein carbonic anhydrase 9 (CAIX) after 10 days of treatment in Colo205 xenografts. This upregulation, presumed to be due to increased hypoxia, may precede and then drive the phenotypic changes associated with vessel normalisation that were observed at later time points (Gene expression profiling of tumour tissues led to the identification of 45 human genes that were differentially expressed after vanucizumab treatment, but were not significantly changed by bevacizumab in this study, thereby representing genes that could explain different treatment effects induced by the two antibodies. Differential expression of 20 genes exclusively in the vanucizumab-treated tumours appeared to be regulated by the combined effect of Ang-2 and VEGF-A inhibition. The expression of 25 other genes was also affected by treatment with LC06, potentially indicating that this regulation is driven by the LC06 component of vanucizumab. Many of the genes are known to be involved in angiogenesis, blood vessel development and/or endothelial cell movement. Other genes have been implicated in microtubule rearrangements or other extracellular processes. Several genes had been previously described as being VEGF-A dependent (ex vivo histology, we have demonstrated that treatment of Colo205 xenografts with vanucizumab results in significant antitumour activity associated with a significant reduction in functional tumour vasculature. Gene expression profiling of tumour tissues identified 20 genes that could be specifically attributed to treatment effects of vanucizumab. Targeting both Ang-2 and VEGF with vanucizumab represents a potent antiangiogenic treatment strategy and whose antivascular effects can be monitored in vivo using DCE MRI. Incorporation of susceptibility contrast MRI measurements into clinical imaging protocols may provide additional confirmatory evidence of antiangiogenic response to vanucizumab in human tumours.In conclusion, using multiparametric MRI combined with


Quantification of renal fluid conservation is possible by urine analysis, and the results can indicate dehydration. The present report sought to determine whether this fluid retention correlates with fluid requirements during major abdominal surgeries that have estimated operating times ≥ 2 h.Urine colour, specific weight, osmolality and creatinine concentration were used to calculate a composite “fluid retention index” (FRI) in 97 patients prior to major abdominal surgery. Goal-directed fluid volume optimization, with hydroxyethyl starch supplemented with a background administration of crystalloid fluid, was used.P < 0.02).The median preoperative FRI was 3.0. Fluid retention, considered as present when FRI ≥ 3.5, was found in 37% of the patients. Fluid retention was followed by a significantly larger blood loss , higher haemorrhage rate  and greater need for both colloid  and crystalloid  fluids. Despite the larger blood loss, the total fluid balance was more positive after surgery in the dehydrated patients  contains supplementary material, which is available to authorized users. Fluid therapy is an inherent component in the management of major abdominal surgery. How the kidneys are currently set to excrete or conserve fluid is of importance to the body’s handling of infusion fluids  surgery, urology or gynaecology) with an expected duration of at least 2 h and age ≥ 18 years. Exclusion criteria were American Society of Anesthesiologists class IV, laparoscopic surgery, cardiac arrhythmias, aortic or mitral insufficiency resulting in haemodynamic impairment, severe renal and hepatic failure, or pulmonary disease that prevented a ventilation volume of 7 ml/kg ideal body weight or the use of PEEP. The study was conducted every time that a suitable patient and the research team were available.From 195 patients screened for eligibility, 150 were randomized between the use of oesophageal Doppler and pulse oximetry (Pleth Variability Index) techniques to guide the goal-directed fluid therapy, which influence on postoperative outcome was a primary aim of the study. Randomization was performed by using opaque envelopes prepared from a computerized procedure. There were 4 drop-outs. From the remaining 146 patients, a preoperative urine sample had been obtained from 112. The results from the first half of this trial have been published elsewhere and show that the amount of fluid infused in the two groups was quite similar throughout the study  was guided by stroke volume changes in accordance with published protocols  of tetrastarch, either Venofundin  or Volulyte , over 3–5 min, using a 50-cmwww.hydrationcheck.com . The urine-specific gravity was determined using Multistix®10 SG reagent strips and a Clinitek Status®+ Analyser .The second tube was sent to the certified clinical chemistry laboratory at Linköping University Hospital for analysis of osmolality.As a quality measure, blood was obtained for measurement of the high-sensitivity troponin T (hsTnT) and the N-terminal fragment of B-type natriuretic peptide (NT-proBNP), before surgery and on the first and second mornings after surgery. The haemoglobin (Hb) concentration was measured invasively just before the induction of anaesthesia, at the end of the surgery, and on the first morning after the surgery. The Hb concentration was monitored by pulse oximetry during the surgery .The use of a composite index for fluid retention that is based on several markers of renal water conservation has the benefit of reducing confounding influences, such as diet, disease and medication, which typically change only one of the markers. The urine colour is due to end products arising from the fairly stable breakdown of erythrocytes, and it darkens with progressive dehydration. The specific gravity of the urine also increases with dehydration, as do the creatinine concentration and the osmolality.fluid retention index (FRI). This term is more appropriate than the previously used “dehydration index” because fluid retention may occasionally be due to other causes than dehydration, in particular in hospital patients.The ranges of colour, osmolality and creatinine concentrations have been published for subjects aged 17–69 years, and each range paralleled the specific gravity scale  for the mean of the four scores. An outlier typically raised the SD to > 1.0. The individual scores were then reviewed and any single outlier was omitted, followed by recalculation of the index. The new value was accepted if SD ≤ 1.0, whereas the index was discarded as being inconclusive if the SD was still > 1.0  − (blood loss + urine).fluid balance in blood volume equivalents was calculated by dividing the crystalloid and the urine volumes by 3 before entering the figures into this equation.The U test or the chi2 test, as appropriate. Correlations between parameters were evaluated by simple and multiple linear regression analysis. P values < 0.05 were considered significant.Continuous and categorical demographic, perioperative and biochemical data were compared using analysis of variance (ANOVA), the Mann-Whitney Preoperative urine samples for analysis of FRI were obtained from 112 patients. The results were incomplete in 6 and inconclusive in 9 cases see the “” sectionThe median (IQR) of the FRI was 3.0 (2.2–3.8) for those 97 patients.P < 0.008). There was no indication that dehydrated patients were less well before the surgery.FRI varied depending on the type of scheduled surgery: gynaecological 2.3 (1.5–3.5), upper GI surgery 3.3 (2.5–3.8), lower GI surgery 3.0 (1.8–4.1) and urological surgery 3.4 (2.5–4.0) , blood loss per operating hour (+ 41%), need for colloid fluid (+ 43%), positive fluid balance (+ 27%) and greater positive fluid balance when expressed in blood volume equivalents , blood loss/operating time (P < 0.03), total fluid balance (P < 0.03) and the fluid balance in blood units (P < 0.02) (two-way ANOVA based on logarithm-transformed data). The real data for all patients is shown in Table Even when controlling for the type of surgery, FRI ≥ 3.5 was still significantly associated with greater blood loss (s P < 0.0 (two-wayNine patients 15%) of the patients with FRI < 3.5 were treated with diuretics on a daily basis (treatment withdrawn on the day of the operation) while 6 received such treatment in the other group (17%). Other chronic medications are listed in Additional file 5% of theFour out of the 14 patients undergoing pancreatectomy including Whipple’s procedure had preoperative serum bilirubin levels above the reference value. Two of them had FRI < 3.5 and two had ≥ 3.5.r = 0.35, P < 0.001), which was due to the gynaecological and urological surgeries , while this relationship was not statistically significant for the GI operations alone . Again, the linear relationship was mostly due to the gynaecological and urological surgeries , while this relationship was not statistically significant for the GI surgeries alone , which was due to the gynaecological and urological surgeries , while this relationship was not statistically significant for the GI surgeries alone  ng/l and the NT-proBNP was 145 (57–250) ng/l.No increase in troponin T occurred in patients with a high FRI, but the NT-proBNP doubled regardless of whether the FRI had a low or high value. Moreover, there were no differences in B-Hb concentration between the groups Table .In a multiple regression model, only the FRI value, but not the ASA class or the mode of monitoring fluid responsiveness , served as a statistically significant predictor of the total fluid balance.Similarly, only the FRI value was a significant predictor of the surgical blood loss.The result confirms that fluid retention, expressed as a high FRI, is associated with a greater need for fluid during surgery to maintain the patients in a fluid optimized state using either oesophageal Doppler or the Pleth Variability Index. The most plausible explanation to this finding is that fluid retention indicated preoperative dehydration, which had to be overcome by infusing larger volumes of fluid.In contrast, the relationship between blood loss and FRI was a surprising post hoc finding. The explanation is unclear, but infusion of a small volume of Ringer’s acetate in these preoperative patients showed that the plasma dilution was greater and the half-life of the fluid twice as long in those with FRI ≥ 3.5  and the GDT optimization as given in this trial resulted in a positive fluid balance of between 1.5 and 2 l. When expressed in blood units, the positive balance averaged 1.1 l, despite a blood loss that was only one third as large. Hence, the recommended “zero balance” (Brandstrup et al. The positive fluid balance was about 1 l when the urine was not concentrated, while twice as large a volume was needed when the urine was concentrated to a degree consistent with dehydration Fig. b.Several mechanisms may explain why a positive fluid balance was required to maintain the central blood flow. Vasodilatation caused by the anaesthesia, as well as transudation into injured tissue, remains the best acknowledged reasons. Fluid retention oedema becomes more pronounced when crystalloid fluid is infused sometime after hydroxyethyl starch (Hahn et al. Despite the administration of larger volumes of fluid, the rise in NT-proBNP was not greater in patients with concentrated urine Table . The aveLimitations of this study were the inclusion of different types of operations, which restricted the possibility of performing statistics. Major surgery is a very complex medical situation, and the mechanisms that govern various parameters, such as urinary excretion, require simpler situations to be readily understood. Among the benefits of the present study is that it was conducted in a standardized way and consistently relied on modern approaches to guide the fluid therapy, although there may be concerns that the oesophageal Doppler and pulse oximetry may not indicate fluid responsiveness correctly.The algorithm used to calculate FRI is based on assessment of urine colour, specific gravity, osmolality and creatinine, which represent metabolic waste products that appear in higher concentrations when the kidneys conserve fluid. The adopted exclusion process ensured that the finally reported FRI values were robust measures of fluid retention. For example, isolated darkening of the urine due to a catheter-induced haematuria or a high serum bilirubin concentration was not allowed to affect the result.Preoperative fluid retention, as given by a urine sample indicating dehydration, was associated with greater fluid requirements during major open abdominal surgery. Fluid retention was also followed by greater blood loss and a need for a more positive fluid balance. These effects were most apparent in gynaecological and urological operations. These results suggest that more fluid should routinely be prescribed to those patients who have concentrated urine before an operation. However, more studies are first needed to determine if more fluid given for this reason result in any benefit for the outcome of the patients.


Non-human primates participating in neurophysiological research are exposed to potentially stressful experimental procedures, such as dietary control protocols, surgical implants and their maintenance, or social separation during training and experimental session. Here, we investigated the effect of controlled access to fluid, surgical implants, implant-related cleaning of skin margins, and behavioral training sessions on salivary cortisol levels of adult male rhesus macaques participating in neurophysiological research. The animals were trained to chew flavored cotton swabs to non-invasively collect saliva samples. Our data show no differences in cortisol levels between animals with and without implants, but both, controlled access to fluid and cleaning of implants individually increased salivary cortisol concentrations, while both together did not further increase the concentration. Specifically, before cleaning, individuals with controlled access to fluid had 55% higher cortisol concentrations than individuals with free access to fluid. Under free access to fluid, cortisol concentrations were 27% higher after cleaning while no effect of cleaning was found for individuals under controlled fluid access. Training sessions under controlled access to fluid also did not affect salivary cortisol concentrations. The observed changes in cortisol concentrations represent mild stress responses, as they are only a fraction of the range of the regular circadian changes in cortisol levels in rhesus monkeys. They also indicate that combinations of procedures do not necessarily lead to cumulative stress responses. Our results indicate that salivary cortisol levels of rhesus monkeys respond to neurophysiological experimental procedures and, hence, may be used to assess further refinements of such experimental methods. Phylogenetic closeness to humans and similarities in physiology, neuroanatomy, cognition and social complexity make non-human primates (NHPs) a vital research model for neuroscience research ,2. Yet, Neurophysiological research has taken considerable strides to improve the welfare of NHPs in research environments, for instance, by adopting behavioral management practices such as social housing, monitoring of physiological parameters, behavioral observations, and positive reinforcement training (PRT). Despite improvements made, neurophysiological research still requires procedures which likely cause stress and raise welfare concerns. For instance, neurophysiological research typically involves separation of an animal from its social group during scientific procedures, movement restraints (by use of primate chairs), surgical implants including their regular cleaning, and fluid or food control protocols to boost the animal’s motivation to participate in training and recording sessions. These procedures likely cause some level of stress or discomfort to the animals, but the extent and whether stress responses to the application of multiple procedures accumulate, remains unclear.Macaca mulatta) have been reported to increase due to social separation )Cortisol levels in animals with and without chronic implants did not differ. Both, controlled fluid regime and cleaning of implants increased salivary cortisol levels, but both together did not further increase the level. Specifically, before cleaning, rhesus macaques under fluid control had 54.7% higher cortisol concentration than individuals with free access to fluid. Under free access to fluid, animals showed an effect of cleaning, with 27.1% higher cortisol concentrations after vs. before implant cleaning. After cleaning, cortisol levels in monkeys with controlled vs. free access to fluid were not significantly different. Also, animals under fluid control showed no difference between before and after cleaning, or between before and after training. This lacking further increase in cortisol in response to implant cleaning or training of fluid-controlled animals is unlikely to be due to a lack in statistical power since there is not even a trend towards higher values compared to the values before implant cleaning .M. fascicularis [The observed moderate increases in cortisol concentration can be considered mild stress responses. First, the effects of experimental conditions on cortisol concentration we observed fell in the lower range of reported effects other stressors cause on cortisol. For instance, the relocation of rhesus macaques to a new and unknown environment without prior training, resulted in an increase in cortisol concentration ranging between 166.0%  and 180.icularis . The sepicularis  or 3 h [icularis  lead to icularis .Cortisol levels did not add for combinations of procedures, indicating no accumulation of stress responses. We did not observe an additive effect between controlled access to fluid and implant cleaning or training, respectively. This lack of additivity cannot be explained by a ceiling effect. For example, the high circadian fluctuation in cortisol level  compareThe way sample were collected and experimental procedures applied did not interfere with the cortisol measurement itself. One might argue that controlling the monkeys’ access to fluid affects their saliva flow rate and, subsequently, affects the measured salivary cortisol concentration. However, neither the increase of saliva flow by administration of citric acid to the tongue nor its decrease with anticholinergic drugs (‘dry mouth’) significantly influenced the salivary cortisol concentration in previous studies Click here for additional data file.S1 TableMean cortisol values (ln-transformed) per animal and condition.(PDF)Click here for additional data file.


Macrophages are conventionally classified into M1 and M2 subtypes according to their differentiation status and functional role in the immune system. However, accumulating evidence suggests that this binary classification system is insufficient to account for the remarkable plasticity of macrophages that gives rise to an immense diversity of subtypes. This diverse spectrum of macrophage subtypes play critical roles in various homeostatic and immune functions, but remain far from being fully characterised. In addition to their roles in normal physiological conditions, macrophages also play crucial roles in disease conditions such as cancer. In this review, we discuss the roles tumour-associated macrophages (TAMs) play in regulating different steps of tumour progression and metastasis, and the opportunities to target them in the quest for cancer prevention and treatment. Macrophages constitute a prominent set of immune cells that are phagocytic in nature and are present in almost all tissues. In general, they are differentiated cells of mononuclear origin and display specific phenotypic characteristics. In mice, macrophages show surface expression of markers such as CD11b, F4/80 and colony-stimulating factor-1 receptor (CSF-1R) and do not express Gr1; whereas, in humans, macrophages show expression of CD68, CD163, CD16, CD312 and CD115 .Macrophages are an incredibly diverse set of cells, constantly altering their functional state in response to environmental stimuli. They undergo the ‘polarisation’ process wherein they express different surface markers and functional programs in response to microenvironmental stimuli such as the cytokines and other signalling mediators. According to the binary polarisation concept, there exist two polarisation states of macrophages: Classically activated macrophages (M1) produce pro-inflammatory cytokines and reactive oxygen/nitrogen species, which are crucial for host defence and tumour cell killing and, therefore, are considered as ‘good’ macrophages. The alternatively activated macrophages (M2) produce anti-inflammatory cytokines and are involved in the resolution of inflammation. These are considered ‘bad’ macrophages because they not only suppress the destructive immunity against parasites and tumour cells, but also promote angiogenesis and matrix remodelling, which make the tumour microenvironment conducive to tumour progression and metastasis .Macrophages not only perform vital functions in normal physiological conditions such as development, wound healing, infection and maintenance of tissue homeostasis, but are also involved in a variety of disease conditions such as autoimmune disorders, atherosclerosis and tumourigenesis , not only reduced macrophage infiltration but also completely abolished tumour progression and metastasis. On the contrary, overexpression of this CSF-1 protein increased the rate of tumour progression and metastasis , thereby negatively regulating their phagocytic activity against tumour cells. Blockade of this PD-1/PD-L1 axis restores phagocytic activity by these TAMs, reduces tumour growth and lengthens survival of mice, strongly suggesting a pro-tumourigenic potential of these TAMs , CD68, and CD80 and CD86 costimulatory molecules.Arg1), MMR (Mrc1), resistin-like molecule α (FIZZ1), and chitinase-like protein Ym1 , and chemokines, and are involved in tissue remodelling and tumour progression  is highly expressed on and is reported as a marker of one macrophage subpopulation found in bone marrow, lymph node, liver, and spleen. Although the information about the signalling pathway involved in the activation of CD169+ macrophages is imprecise, CD169+ macrophages are mainly involved in erythropoiesis and immune regulation , promote proteolytic remodelling of the extracellular matrix, accelerate tumour motility and induce migration and invasion of tumour cells . AccordiThe tumour microenvironment is a complex ecology of heterogeneous cell populations, which have a robust influence on tumourigenesis. For example, TAM–adipocyte interactions have been shown to drive cancer initiation and progression in case of obesity and overweight conditions. Adipocyte hypertrophy, inflammation and apoptosis results in macrophage recruitment, which phagocytose dead/dying adipocytes and develop inflammatory foci called crown-like structures (CLS). Many reports have shown that these adipocytes and apoptotic CLSs promote tumour progression in breast . Recentlin vitro, suggesting the pro-angiogenic action of TAM-derived ADM , prevented macrophage entry into the hypoxic region and ablated pro-angiogenic and immunosuppressive functions of TAMs, thereby inhibiting tumour growth and metastasis , endothelin, eotaxin and oncostatin M. Once macrophages arrive in these tumour compartments, their migration is halted via hypoxia-dependent mechanisms. Pro-tumoural functions of macrophages are then facilitated by a hypoxia-dependent transcription factor HIF1a, which induces expression of a large set of genes associated with angiogenesis such as VEGF . This prin vivo, and also markedly enhanced the invasiveness of cancer cells in culture , ZEB2, and α-smooth muscle actin, as a result of which the cells acquire the ability to migrate and invade, leading to tumour progression and metastasis. Regulation of EMT is mediated by many growth factors and cytokines such as TGF-β, forkhead box protein M1 (FoxM1), hepatocyte growth factor, EGF, NFκB, Notch, and Wnt , which is secreted by hypoxic breast tumour cells and is a major target of hypoxia-inducible factor (HIF) signalling, stabilises the ECM network by cross-linking collagen IV in the basement membranes at the pre-metastatic sites. This crosslinking facilitates myeloid cell recruitment and subsequent tumour cell colonisation. Moreover, LOX ablation prevents the formation of such sites and inhibits metastatic growth  to form a premetastatic niche that ultimately promoted liver metastases -derived cell types, increased numbers of fibroblasts, secreted oncoproteins, and cytokines. Kaplan et al showed a positive correlation between infiltration of macrophages and glioma initiating cancer stem cells or GICSCs. TAMs were localised in higher densities in areas with a higher number of GICSCs with close contacts between the two cell types. Moreover, in glioma tissue, the secretion of CSC-derived chemoattractants such as CCL2, CCL5, VEGF-A was much higher, facilitating recruitment of macrophages  and IL-6 through coordinated activation of the STAT3 and sonic hedgehog pathways. Interestingly, CSC population is the major population promoting the production of MFG-E8 and IL-6 from macrophages, suggesting that they impart macrophages with the ability to produce tumourigenic factors such as MFG-E8 and IL-6  and cytotoxic T-lymphocyte antigen 4 (CTLA4), respectively. Activation of these receptors by their ligands results in inhibition of TCR signalling and T-cell cytotoxic function . MoreoveSeveral reports have provided strong evidence that TAMs are crucial components of the tumour microenvironment and TAM infiltration is strongly associated with poor prognosis and survival rates in cancer patients. Based on these findings, targeting TAMs is emerging as an attractive strategy for therapeutic intervention .It is now well-established that chemoattractants in tumour microenvironment facilitate massive infiltration of macrophages in tumours. Hence, depletion or inhibition of TAM recruitment by modulating levels of these chemoattractants may serve as an effective strategy. Indeed, pharmacological inhibition, neutralising monoclonal antibodies, or genetic mutation of chemoattractants such as CCL2, VEGFR2, CSF-1R depleted TAM infiltration and reduced tumour growth. For instance, in case of CSF1R, the humanised monoclonal antibody RG7155 potently inhibited CSF1R dimerisation and also induced a striking reduction in the CSF1R+CD163+ macrophage population within tumour tissues (in vivo (Differentiation of pro-tumourigenic M2 to the anti-tumour M1 phenotype is rapidly emerging as a new therapeutic approach. Activation of TLR3/Toll-IL-1 receptor domain-containing adaptor molecule 1 by Poly (I:C) rapidly enhanced secretion of pro-inflammatory cytokines and accelerated M1 macrophage polarisation (The conventional binary model of macrophage polarisation is becoming antiquated and a newer model of polarisation spectrum is coming into conception, which focuses on involvement of an array of differentiated macrophages in various immunoregulatory disorders as well as cancers. Moreover, it is now well-established that apart from tumour cell-derived factors, stromal microenvironmental factors play a substantial role in tumourigenesis and TAMs constitute as key players in this phenomenon by regulating various steps of tumour initiation, progression, and metastasis. Since the abrogation of immunosuppressive macrophages in the tumour microenvironment enhances anti-tumour response, targeting TAMs is rapidly emerging as a promising therapeutic strategy for cancer patients.