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Molecular imaging technologies are increasingly used to diagnose, monitor, and guide treatment of i.e., cancer. In this review, the current status and future prospects of the use of molecular imaging as an instrument to help realize precision surgery is addressed with focus on the main components that form the conceptual basis of intraoperative molecular imaging. Paramount for successful interventions is the relevance and accessibility of surgical targets. In addition, selection of the correct combination of imaging agents and modalities is critical to visualize both microscopic and bulk disease sites with high affinity and specificity. In this context developments within engineering/imaging physics continue to drive the growth of image-guided surgery. Particularly important herein is enhancement of sensitivity through improved contrast and spatial resolution, features that are critical if sites of cancer involvement are not to be overlooked during surgery. By facilitating the connection between surgical planning and surgical execution, digital surgery technologies such as computer-aided visualization nicely complement these technologies. The complexity of image guidance, combined with the plurality of technologies that are becoming available, also drives the need for evaluation mechanisms that can objectively score the impact that technologies exert on the performance of healthcare professionals and outcome improvement for patients.

Purpose Sentinel lymph node biopsy is a routine procedure for nodal staging in penile cancer. Most commonly, this procedure is guided by radioactive tracers, providing various forms of preoperative and intraoperative guidance. This is further extended with fluorescence imaging using hybrid radioactive–fluorescence tracers. Alternatively, a magnetic-based approach has become available using superparamagnetic iron-oxide nanoparticles (SPIONs). This study investigates a novel freehand magnetic particle imaging and navigation modality (fhMPI) for intraoperative localization, along with a hybrid approach, combining magnetic and fluorescence guidance. Materials and methods The fhMPI set-up was built with a surgical navigation device, optical tracking system and magnetometer probe. A dedicated reconstruction software based on a look-up-table method was used to reconstruct a superficial 3D volume of the SPION distribution in tissue. For fluorescence guidance, indocyanine green (ICG) was added to the SPIONs. The fhMPI modality was characterized in phantoms, ex vivo human skin and in vivo porcine surgery. Results Phantom and human skin explants illustrated that the current fhMPI modality had a sensitivity of 2.2 × 10–2 mg/mL SPIONs, a resolving power of at least 7 mm and a depth penetration up to 1.5 cm. Evaluation during porcine surgery showed that fhMPI allowed for an augmented reality image overlay of the tracer distribution in tissue, as well as 3D virtual navigation. Besides, using the hybrid approach, fluorescence imaging provided a visual confirmation of localized nodes. Conclusion fhMPI is feasible in vivo, providing 3D imaging and navigation for magnetic nanoparticles in the operating room, expanding the guidance possibilities during magnetic sentinel lymph node procedures. Furthermore, the integration of ICG provides the ability to visually refine and confirm correct localization. Further clinical evaluation should verify these findings in human patients as well.

Background: Sentinel lymph node (SN) biopsy (SLNB) has proven to be a valuable tool for staging melanoma patients. Since its introduction in the early 1990s, this procedure has undergone several technological refinements, including the introduction of single photon emission computed tomography combined with computed tomography (SPECT/CT) as well as radio- and fluorescence-guidance. The purpose of the current study was to evaluate the effect of this technological evolution on SLNB in the head and neck region. Primary endpoint was the false-negative (FN) rate. Secondary endpoints were number of harvested SNs, overall operation time, operation time per harvested SN and postoperative complications. Patients and Methods: A retrospective database was queried for cutaneous head and neck melanoma patients who underwent SLNB at The Netherlands Cancer Institute between 1993 and 2016. The implementation of new detection techniques was divided in 4 groups: (1) 1993-2005, with preoperative lymphoscintigraphy and intraoperative use of both a gamma ray detection probe and patent blue (n = 30); (2) 2006-2007, with addition of preoperative roadmaps based on SPECT/CT (n = 15); (3) 2008-2009, with intraoperative use of a portable gamma camera (n = 40); and (4) 2010-2016, with the addition of near-infrared fluorescence guidance (n = 192). Results: A total of 277 patients were included. At least one SN was identified in all patients. A tumor-positive SN was found in 59 patients (21.3%), 10 in group 1 (33.3%), 3 in group 2 (20.0%), 6 in group 3 (15.0%) and 40 in group 4 (20.8%). Regional recurrences of patients with tumor-negative SNs resulted in an overall FN rate of 11.9% (FN groups 1: 16.7%; 2: 0%; 3: 14.3%; 4: 11.1%). The number of harvested nodes increased with advancing technologies (P = 0.003) whereas Breslow thickness and operation time per harvested SN decreased (P = 0.003 and P = 0.017, respectively). There was no significant difference in percentage of tumor-positive SNs, overall operation time and complication rate between the different groups. Conclusion: The use of advanced detection technologies led to a higher number of identified SNs without increase in overall operation time, which may indicate an improved surgical efficiency. Operation time per harvested SN decreased, the average FN rate remained 11.9% and unchanged over 23 years. There was no significant change in postoperative complication rate.

The increasing availability of new imaging technologies and tracers has enhanced the application of nuclear molecular imaging in urogenital interventions. In this context, preoperative nuclear imaging and radioactivity-based intraoperative surgical guidance have become important tools for the identification and anatomical allocation of tumor lesions and/or suspected lymph nodes. Fluorescence guidance can provide visual identification of the preoperatively defined lesions during surgery. However, the added value of fluorescence guidance is still mostly unknown. This review provides an overview of the role of fluorescence imaging in radioguided surgery in urogenital malignancies. The sentinel node (SN) biopsy procedure using hybrid tracers (radioactive and fluorescent component) serves as a prominent example for in-depth evaluation of the complementary value of radio- and fluorescence guidance. The first large patient cohort and long-term follow-up studies show: 1) improvement in the SN identification rate compared to blue dye; 2) improved detection of cancer-positive SNs; and 3) hints towards a positive effect on (biochemical) recurrence rates compared to extended lymph node dissection. The hybrid tracer approach also highlights the necessity of a preoperative roadmap in preventing incomplete resection. Recent developments focus on receptor-targeted approaches that allow intraoperative identification of tumor tissue. Here radioguidance is still leading, but fluorescent and hybrid tracers are also finding their way into the clinic. Emerging multiwavelength approaches that allow concomitant visualization of different anatomical features within the surgical field may provide the next step towards even more refined procedures.

Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, (99)mTc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation (99)mTc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Methods: Several PSMA-targeted hybrid tracers were synthesized: glutamic acid- urea-lysine (EuK)-Cy5-mas(3), EuK-(SO3)Cy5-mas(3), EuK-Cy5(SO3)-mas(3), EuK-(Ar)Cy5-mas(3), and EuK-Cy5(Ar)-mas(3); the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas(3)) chelate to study the dye's interaction with PSMA's amphipathic entrance funnel. The compounds were evaluated for their photophysical and chemical properties and PSMA affinity. After radiolabeling with (99)mTc, we performed in vivo SPECT imaging, biodistribution, and fluorescence imaging on BALB/c nude mice with orthotopically transplanted PC346C tumors. Results: The dye composition influenced the photophysical properties (brightness range 0.3-1.5 x 10(4) M-1 x cm(-1)), plasma protein interactions (range 85.0% +/- 2.3%-90.7%+/- 1.3% bound to serum, range 76%+/- 0%-89%+/- 6% stability in serum), PSMA affinity (half-maximal inhibitory concentration [IC50] range 19.2 +/- 5.8-175.3 +/- 61.1 nM) and in vivo characteristics (tumorto-prostate and tumor-to-muscle ratios range 0.02 +/- 0.00-154.73 +/- 28.48 and 0.46 +/- 0.28-5,157.50 +/- 949.17, respectively; renal, splenic, and salivary retention). Even though all tracer analogs allowed tumor identification with SPECT and fluorescence imaging, (99)mTc-EuK(SO3)Cy5-mas(3) had the most promising properties (e.g., half-maximal inhibitory concentration, 19.2 +/- 5.8, tumor-to-muscle ratio, 5,157.50 +/- 949.17). Conclusion: Our findings demonstrate the intrinsic integration of a fluorophore in the pharmacophore in PSMA-targeted smallmolecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK ((99)mTc-EuK-(SO3)Cy5-mas(3)) yielded the most promising tracer candidate for imaging of PSMA.

Previous studies indicated that location and amount of detected sentinel lymph nodes (SLNs) in prostate cancer (PCa) are influenced where SLN-tracer is deposited within the prostate. To validate whether intratumoral (IT) tracer injection helps to increase identification of tumor-positive lymph nodes (LNs) better than intraprostatic (IP) tracer injection, a prospective randomized phase II trial was performed.PCa patients with a 5% risk of lymphatic involvement were randomized between ultrasound-guided transrectal injection of indocyanine green-[IT-injection did not result in significant difference of removed SLNs (5.0 vs 6.0, p = 0.317) and histologically positive SLNs (28 vs 22, p = 0.571). However, in IT-group, the SLN-positive nodes were 73.7% of total positive nodes compared to 37.3% in IP-group (p = 0.015). Moreover, significantly more node-positive patients were found in IT-group (42% vs 24%, p = 0.045), which did not result in worse MFS. In two patients (3.6%) from whom the IT-tracer injection only partly covered intraprostatic tumor spread, nodal metastases in ePLND without tumor-positive SNs were yielded.The percentage-positive SLNs found after IT-injection were significantly higher compared to IP-injection. Significantly more node-positive patients were found using IT-injection, which did not affect MFS. IT-injection failed to detect nodal metastases from non-index satellite lesions. Therefore, we suggest to combine IT- and IP-tracer injections in men with visible tumor on imaging.

Within interventional nuclear medicine (iNM) a prominent role is allocated for the sub-discipline of radioguided surgery. Unique for this discipline is the fact that an increasing number of clinical indications (e.g. lymphatic mapping, local tumor demarcation and/or tumor receptor targeted applications) have been adopted into routine care. The clinical integration is further strengthened by technical innovations in chemistry and engineering that enhance the translational potential of radioguided procedures in iNM. Together, these features not only ensure ongoing expansion of iNM but also warrant a lasting clinical impact for the sub-discipline of radioguided surgery.

The field of fluorescence-guided surgery builds on colored fluorescent tracers that have become available for different clinical applications. Combined use of complementary fluorescent emissions can allow visualization of different anatomical structures (e.g. tumor, lymphatics and nerves) in the same patient. With the aim to assess the requirements for multi-color fluorescence guidance under in vivo conditions, we thoroughly characterized two FDA-approved laparoscopic Firefly camera systems available on the da Vinci Si or da Vinci Xi surgical robot. In this process, we studied the cameras’ performance with respect to the photophysical properties of the FDA-approved dyes Fluorescein and ICG. Our findings indicate that multi-wavelength fluorescence imaging of Fluorescein and ICG is possible using clinical-grade fluorescence laparoscopes, but critical factors for success include the photophysical dye properties, imaging system performance and the amount of accumulated dye. When comparing the camera performance, the Xi system provided more effective excitation (adaptions in the light source) and higher detection sensitivity (chip-on-a-tip and/or enhanced image processing) for both Fluorescein and ICG. Both systems can readily be used for multi-wavelength fluorescence imaging of Fluorescein and ICG under clinically relevant conditions. With that, another step has been made towards the routine implementation of multi-wavelength image-guided surgery concepts.

Tracers can help visualize the lymphatic drainage patterns and sentinel nodes (SNs) of individual prostate cancer patients. To determine the role of nuclear medicine in surgical guidance, in particular the positional guidance of a SPECT/CT-based 3-dimensional imaging road map, in this process we studied to what extent fluorescence guidance underestimated the number of target lesions relative to radioguidance. Methods: SPECT/CT imaging was performed after intraprostatic tracer administration of either indocyanine green (ICG)-Tc-99m-nanocolloid (hybrid-tracer group) or Tc-99m-nanocolloid to create a road map that depicted all SNs. Patients who received Tc-99m-nanocolloid were injected with "free" ICG immediately before surgery. Before unmasking, fluorescence guidance was used for intraoperative SN identification. This was followed by extended pelvic lymph node dissection (ePLND). After unmasking of the SPECT/CT images, the number of missed SNs was recorded and their resection was pursued when there was no risk of intraoperative complications. Results: Preoperative SPECT/CT revealed no differences in the SN identification rate between ICG-Tc-99m-nanocolloid and Tc-99m-nanocolloid. However, fluorescence guidance allowed intraoperative removal of all SNs in only 40% of patients in the hybrid-tracer group and 20% of patients in the free-ICG group. Overall, 75.9% of the intraoperatively resected SNs in the hybrid-tracer group and 51.8% of the SNs in the free-ICG group were removed solely under fluorescence guidance. During ePLND, 22 additional SNs were resected (7 in the hybrid-tracer group and 15 in the free-ICG group). After unmasking, 18 remaining SNs were identified (6 in the hybrid group and 12 in the free-ICG group). In the free-ICG group, ex vivo evaluation of the excised specimens revealed that 14 SNs removed under ePLND or after unmasking contained radioactivity but no fluorescence. Conclusion: The preoperative imaging road map provided by SPECT/CT enhanced the detection of prostate SNs in more ectopic locations in 17 of the 25 patients, and the hybrid tracer ICG-Tc-99m-nanocolloid was shown to outperform free ICG. Overall, fluorescence-guided pelvic nodal surgery underestimated the number of SNs in 60%-80% of patients.

Background During (robot-assisted) sentinel node (SN) biopsy procedures, intraoperative fluorescence imaging can be used to enhance radioguided SN excision. For this combined pre- and intraoperative SN identification was realized using the hybrid SN tracer, indocyanine green- 99m Tc-nanocolloid. Combining this dedicated SN tracer with a lymphangiographic tracer such as fluorescein may further enhance the accuracy of SN biopsy. Objective Clinical evaluation of a multispectral fluorescence guided surgery approach using the dedicated SN tracer ICG- 99m Tc-nanocolloid, the lymphangiographic tracer fluorescein, and a commercially available fluorescence laparoscope. Design, setting, and participants Pilot study in ten patients with prostate cancer. Following ICG- 99m Tc-nanocolloid administration and preoperative lymphoscintigraphy and single-photon emission computed tomograpy imaging, the number and location of SNs were determined. Fluorescein was injected intraprostatically immediately after the patient was anesthetized. A multispectral fluorescence laparoscope was used intraoperatively to identify both fluorescent signatures. Surgical procedure Multispectral fluorescence imaging during robot-assisted radical prostatectomy with extended pelvic lymph node dissection and SN biopsy. Measurements ( 1) Number and location of preoperatively identified SNs. (2) Number and location of SNs intraoperatively identified via ICG- 99m Tc-nanocolloid imaging. (3) Rate of intraoperative lymphatic duct identification via fluorescein imaging. (4) Tumor status of excised (sentinel) lymph node(s). (5) Postoperative complications and follow-up. Results and limitations Near-infrared fluorescence imaging of ICG- 99m Tc-nanocolloid visualized 85.3% of the SNs. In 8/10 patients, fluorescein imaging allowed bright and accurate identification of lymphatic ducts, although higher background staining and tracer washout were observed. The main limitation is the small patient population. Conclusion Our findings indicate that a lymphangiographic tracer can provide additional information during SN biopsy based on ICG- 99m Tc-nanocolloid. The study suggests that multispectral fluorescence image–guided surgery is clinically feasible. Patient summary We evaluated the concept of surgical fluorescence guidance using differently colored dyes that visualize complementary features. In the future this concept may provide better guidance towards diseased tissue while sparing healthy tissue, and could thus improve functional and oncologic outcomes.

By contributing to noninvasive molecular imaging and radioguided surgery, nuclear medicine has been instrumental in the realization of precision medicine. During the last decade, it has also become apparent that nuclear medicine (e.g., in the form of bimodal/hybrid tracers) can help to empower fluorescence-guided surgery. More specifically, when using hybrid tracers, lesions can be noninvasively identified and localized with a high sensitivity and precision (guided by the radioisotope) and ultimately resected under real-time optical guidance (fluorescent dye). This topical review discusses early clinical successes, preclinical directions, and key aspects that could have an impact on the future of this field.

Diagnostic imaging modalities have moderate sensitivity for the identification of lymph node (LN) metastases in prostate cancer (PCa) patients. Mapping the lymphatic drainage from the prostate can help to identify the LNs directly draining from the tumour (sentinel nodes (SNs)); the LNs stated to have the highest chance of containing metastatic cancer cells. Although the lymphatic drainage may differ between segments within the prostate, the location of the primary tumour is not routinely taken into account during peripheral zone-aimed tracer administration. This study evaluates whether linking the SN procedure to the primary cancer deposits increases the identification accuracy of lymphatic metastases. Sixty-seven PCa patients, scheduled for robot-assisted laparoscopic prostatectomy (RALP) and extended lymph node dissection (ePLND) with subsequent SN biopsy, were included in this retrospective study. After injection of the hybrid tracer ICG-99mTc-nanocolloid in the prostate, SN mapping was performed based on lymphoscintigraphy and SPECT/CT. SNs were resected using a combination of radio- and fluorescence guidance. Pathology was used to determine the primary tumour location and metastatic spread. Fluorescence imaging of paraffin-embedded prostate tissue was used to determine the location of the tracer deposits in the prostate. This deposition was related to the primary tumour location, the lymphatic drainage pattern of the injected tracer, and the metastatic spread. In total 265 radioactive LNs (211 SNs and 54 higher-echelon nodes in 64 patients; 4.3 LNs per patient; IQR: 2–6) were identified. In three patients (4%) preoperative imaging did not allow identification of SNs. Tumour-positive SN visualization within the pelvis was shown to be influenced by intraprostatic location of tracer administration. This could be concluded from (1) a clear correlation between lymphatic drainage to the right or left side of the body and tracer deposition on the right or left side of the prostate, (2) visualization of a higher number of LNs after dorsal tracer deposition compared with ventral tracer deposition, (3) different drainage patterns observed for tracer deposition into the base or apex of the prostate, and (4) the indication that intratumoural tracer deposition increases the chance of visualizing nodal metastases compared with extratumoural tracer deposition. The correlation between the location of the tracer deposits, the location of the primary tumour, and the visualization of the (tumour-positive) SNs indicated that placement of tracer deposits is of influence on the visualized lymphatic drainage pattern. This suggests that tracer injection near or into the primary tumour site is beneficial for the identification of metastatic spread.

This work evaluates the possibility of placement of high-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) within precision medicine by assessing the suitability of LA-ICP-MS as a micro-analytical technique for the localization and quantification of membranous receptors in heterogeneous cell samples that express both the membrane-bound receptors C-X-C chemokine receptor type 4 (CXCR4) and epidermal growth factor receptor (EGFR). Staining of the breast cancer cell lines MDA-MB-231 X4 and MDA-MB-468 was achieved using receptor-specific hybrid tracers, containing both a fluorophore and a DTPA single-lanthanide chelate. Prior to LA-ICP-MS imaging, fluorescence confocal microscopy (FCM) imaging was performed to localize the receptors, hereby enabling direct comparison. Based on the different expression levels of CXCR4 and EGFR, a distinction could be made between the cell lines using both imaging modalities. Furthermore, FCM and LA-ICP-MS demonstrated complementary characteristics, as a more distinct discrimination could be made between both cell lines based on the EGFR-targeting hybrid tracer via LA-ICP-MS, due to the intrinsic CXCR4-related green fluorescent protein (GFP) signal present in the MDA-MB-231 X4 cells. Employing state-of-the-art LA-ICP-MS instrumentation in bidirectional area scanning mode for sub-cellular imaging of MDA-MB-231 X4 cells enabled the specific binding of the CXCR4-targeting hybrid tracer to the cell membrane to be clearly demonstrated. The stretching of cells over the glass substrate led to a considerably higher signal response for pixels at the cell edges, relative to the more central pixels. The determination of the expression levels of CXCR4 and EGFR for the MDA-MB-468 cell line was performed using LA-ICP-MS single-cell analysis (sc-LA-ICP-MS) and external calibration, based on the quantitative ablation of Ho-spiked dried gelatin droplet standards. Additionally, a second calibration approach was applied based on spot ablation of highly homogeneous dried gelatin gels in combination with the determination of the ablated volume using atomic force microscopy (AFM) and yielded results which were in good agreement with the expression levels determined via flow cytometry (FC) and mass cytometry (MC). Hybrid tracers enable a direct comparison between (i) FCM and LA-ICP-MS imaging for the evaluation of the microscopic binding pattern and between (ii) FC, MC and sc-LA-ICP-MS for the quantification of receptor expression levels in single cells.

The aim of the current narrative review was to summarize the available evidence in the literature on artificial intelligence (AI) methods that have been applied during robotic surgery.A narrative review of the literature was performed on MEDLINE/Pubmed and Scopus database on the topics of artificial intelligence, autonomous surgery, machine learning, robotic surgery, and surgical navigation, focusing on articles published between January 2015 and June 2019. All available evidences were analyzed and summarized herein after an interactive peer-review process of the panel.The preliminary results of the implementation of AI in clinical setting are encouraging. By providing a readout of the full telemetry and a sophisticated viewing console, robot-assisted surgery can be used to study and refine the application of AI in surgical practice. Machine learning approaches strengthen the feedback regarding surgical skills acquisition, efficiency of the surgical process, surgical guidance and prediction of postoperative outcomes. Tension-sensors on the robotic arms and the integration of augmented reality methods can help enhance the surgical experience and monitor organ movements.The use of AI in robotic surgery is expected to have a significant impact on future surgical training as well as enhance the surgical experience during a procedure. Both aim to realize precision surgery and thus to increase the quality of the surgical care. Implementation of AI in master-slave robotic surgery may allow for the careful, step-by-step consideration of autonomous robotic surgery.

Purpose Even when guided by SPECT/CT planning of nodal resection in the head-and-neck area is challenging due to the many critical anatomical structures present within the surgical field. In this study the potential of a (SPECT/)MRI-based surgical planning method was explored. Hereby MRI increases the identification of SNs within clustered lymph nodes (LNs) and vital structures located adjacent to the SN (such as cranial nerve branches). Method and patients SPECT/CT and pathology reports from 100 head-and-neck melanoma and 40 oral cavity cancer patients were retrospectively assessed for SN locations in levels I–V and degree of nodal clustering. A diffusion-weighted-preparation magnetic resonance neurography (MRN) sequence was used in eight healthy volunteers to detect LNs and peripheral nerves. Results In 15% of patients clustered nodes were retrospectively shown to be present at the location where the SN was identified on SPECT/CT (level IIA: 37.2%, level IIB: 21.6% and level III: 15.5%). With MRN, improved LN delineation enabled discrimination of individual LNs within a cluster. Uniquely, this MRI technology also provided insight in LN distribution (23.2 ± 4 LNs per subject) and size (range 21–372 mm3), and enabled non-invasive assessment of anatomical variances in the location of the LNs and facial nerves. Conclusion Diffusion-weighted-preparation MRN enabled improved delineation of LNs and their surrounding delicate anatomical structures in the areas that most often harbor SNs in the head-and-neck. Based on our findings a combined SPECT/MRI approach is envisioned for future surgical planning of complex SN resections in this region.

Summary The impact of a compromised blood-brain barrier (BBB) on the drug treatment of intracranial tumors remains controversial. We characterize the BBB integrity in several intracranial tumor models using magnetic resonance imaging, fluorescent dyes, and autoradiography and determine the distribution and efficacy of docetaxel in brain tumors grafted in Abcb1-proficient and Abcb1-deficient mice. Leakiness of the tumor vasculature varies from extensive to absent. Regardless of the extent of leakiness, tumor blood vessels express ATP-binding cassette transporters (Abcb1 and Abcg2). A leaky vasculature results in higher docetaxel tumor levels compared to normal brain. Nevertheless, Abcb1 can reduce drug distribution and efficacy even in leaky models. Thus, BBB leakiness does not ensure the unimpeded access of ATP-binding cassette transporter substrate drugs. Therapeutic responses may be observed, but the full potential of such therapeutics may still be attenuated. Consequently, BBB-penetrable drugs with little to no affinity for efflux transporters are preferred for the treatment of intracranial tumors., Graphical Abstract, Highlights Blood-brain barrier integrity in brain tumor models varies from intact to absent Brain tumor vessels express drug efflux transporters Drug transporters can impede drug entry and efficacy, even in leaky tumors Low-affinity ABC transporter drugs are favored candidates for treating brain tumors, Whether a compromised blood-brain barrier hinders the drug treatment of intracranial tumors remains controversial. de Gooijer et al. determine how drug transporters affect the efficacy of docetaxel against several intracranial tumor models with various levels of blood-brain barrier integrity and show that ABC transporters can restrict drug efficacy even when tumor vessels are leaky.

Hepatic radioembolization therapies can suffer from discrepancies between diagnostic planning (scout-scan) and the therapeutic delivery itself, resulting in unwanted side-effects such as pulmonary shunting. We reasoned that a nanotechnology-based pre-targeting strategy could help overcome this shortcoming by directly linking pre-interventional diagnostics to the local delivery of therapy. Methods: The host-guest interaction between adamantane and cyclodextrin was employed in an in vivo pre-targeting set-up. Adamantane (guest)-functionalized macro albumin aggregates (MAA-Ad; d = 18 μm) and (radiolabeled) Cy5 and β-cyclodextrin (host)-containing PIBMA polymers (99mTc-Cy50.5CD10PIBMA39; MW ~ 18.8 kDa) functioned as the reactive pair. Following liver or lung embolization with (99mTc)-MAA-Ad or (99mTc)-MAA (controls), the utility of the pre-targeting concept was evaluated after intravenous administration of 99mTc-Cy50.5CD10PIBMA39. Results: Interactions between MAA-Ad and Cy50.5CD10PIBMA39 could be monitored in solution using confocal microscopy and were quantified by radioisotope-based binding experiments. In vivo the accumulation of the MAA-Ad particles in the liver or lungs yielded an approximate ten-fold increase in accumulation of 99mTc-Cy50.5CD10PIBMA39 in those organs (16.2 %ID/g and 10.5 %ID/g, respectively) compared to the control. Pre-targeting with MAA alone was shown to be only half as efficient. Uniquely, for the first time, this data demonstrates that the formation of supramolecular interactions between cyclodextrin and adamantane can be used to drive complex formation in the chemically challenging in vivo environment. Conclusion: The in vivo distribution pattern of the cyclodextrin host could be guided by the pre-administration of the adamantane guest, thereby creating a direct link between the scout-scan (MAA-Ad) and delivery of therapy.

Expressed on virtually all prostate cancers and their metastases, the transmembrane protein prostate-specific membrane antigen (PSMA) provides a valuable target for the imaging of prostate cancer. Not only does PSMA provide a target for noninvasive diagnostic imaging, e.g., PSMA-positron emission tomography (PSMA PET), it can also be used to guide surgical resections of PSMA-positive lesions. The latter characteristic has led to the development of a plethora of PSMA-targeted tracers, i.e., radiolabeled, fluorescent, or hybrid. With image-guided surgery applications in mind, this review discusses these compounds based on clinical need. Here, the focus is on the chemical aspects (e.g., imaging label, spacer moiety, and targeting vector) and their impact on in vitro and in vivo tracer characteristics (e.g., affinity, tumor uptake, and clearance pattern).

Purpose Hybrid image-guided surgery technologies such as combined radio- and fluorescence-guidance are increasingly gaining interest, but their added value still needs to be proven. In order to evaluate if and how fluorescence-guidance can help realize improvements beyond the current state-of-the-art in sentinel node (SN) biopsy procedures, use of the hybrid tracer indocyanine green (ICG)-99mTc-nancolloid was evaluated in a large cohort of patients. Patients and methods A prospective trial was conducted (n = 501 procedures) in a heterogeneous cohort of 495 patients with different malignancies (skin malignancies, oral cavity cancer, penile cancer, prostate cancer and vulva cancer). After injection of ICG-99mTc-nanocolloid, SNs were preoperatively identified based on lymphoscintigraphy and SPECT/CT. Intraoperatively, SNs were pursued via gamma tracing, visual identification (blue dye) and/or near-infrared fluorescence imaging during either open surgical procedures (head and neck, penile, vulvar cancer and melanoma) or robot assisted laparoscopic surgery (prostate cancer). As the patients acted as their own control, use of hybrid guidance could be compared to conventional radioguidance and the use of blue dye (n = 300). This was based on reported surgical complications, overall survival, LN recurrence free survival, and false negative rates (FNR). Results A total of 1,327 SN-related hotspots were identified on 501 preoperative SPECT/CT scans. Intraoperatively, a total number of 1,643 SNs were identified based on the combination of gamma-tracing (>98%) and fluorescence-guidance (>95%). In patients wherein blue dye was used (n = 300) fluorescence-based SN detection was superior over visual blue dye-based detection (22–78%). No adverse effects related to the use of the hybrid tracer or the fluorescence-guidance procedure were found and outcome values were not negatively influenced. Conclusion With ICG-99mTc-nanocolloid, the SN biopsy procedure has become more accurate and independent of the use of blue dye. With that, the procedure has evolved to be universal for different malignancies and anatomical locations. Electronic supplementary material The online version of this article (10.1007/s00259-018-4028-x) contains supplementary material, which is available to authorized users.

The potential of receptor-mediated fluorescence-based image-guided surgery tracers is generally linked to the near-infrared emission profile and good-manufacturing-production availability of fluorescent dyes. Surprisingly, little is known about the critical interaction between the structural composition of the dyes and the pharmacokinetics of the tracers. In this study, a dual-modality tracer design was used to systematically and quantitatively evaluate the influence of elongation of the polymethine chain in a fluorescent cyanine dye on the imaging potential of a targeted tracer. Methods: As a model system, the integrin marker αvβ3 was targeted using arginylglycylaspartisc acid [RGD]-based vectors functionalized with a 111In-diethylenetriaminepentaacetic acid (DTPA) chelate and a fluorescent dye: (Cy3-(SO3)methyl-COOH [emission wavelength (λem), 580 nm], Cy5-(SO3)methyl-COOH [λem, 680 nm], or Cy7-(SO3)methyl-COOH [λem, 780 nm]). Tracers were analyzed for differences in photophysical properties, serum protein binding, chemical or optical stability, and signal penetration through tissue. Receptor affinities were evaluated using saturation and competition experiments. In vivo biodistribution (SPECT imaging and percentage injected dose per gram of tissue) was assessed in tumor-bearing mice and complemented with in vivo and ex vivo fluorescence images obtained using a clinical-grade multispectral fluorescence laparoscope. Results: Two carbon-atom-step variations in the polymethine chain of the fluorescent cyanine dyes were shown to significantly influence the chemical and photophysical characteristics (e.g., stability, brightness, and tissue penetration) of the hybrid RGD tracers. DTPA-Cy5-(SO3)methyl-COOH-c[RGDyK] structurally outperformed its Cy3 and Cy7 derivatives. Radioactivity-based evaluation of in vivo tracer pharmacokinetics yielded the lowest nonspecific uptake and highest tumor-to-background ratio for DTPA-Cy5-(SO3)methyl-COOH-c[RGDyK] (13.2 ± 1.7), with the Cy3 and Cy7 analogs trailing at respective tumor-to-background ratios of 5.7 ± 0.7 and 4.7 ± 0.7. Fluorescence-based assessment of tumor visibility revealed a similar trend. Conclusion: These findings underline that variations in the polymethine chain lengths of cyanine dyes have a profound influence on the photophysical properties, stability, and in vivo targeting capabilities of fluorescent imaging tracers. In a direct comparison, the intermediate-length dye (Cy5) yielded a superior c[RGDyK] tracer, compared with the shorter (Cy3) and longer (Cy7) analogs.

To determine the correlation of preoperative fascia thickness (FT) and intraoperative fascia preservation (FP) with erectile function (EF) after nerve-sparing robot-assisted radical prostatectomy (RARP). Our analysis included 106 patients, with localized prostate cancer and no erectile dysfunction (ED) before RARP, assessed with preoperative 3 Tesla (3 T) multiparametric magnetic resonance imaging (MRI). FP score was defined as the extent of FP from the base to the apex of the prostate, quantitatively assessed by the surgeon. Median fascia thickness (MFT) per patient was defined as the sum of the median FT of 12 MRI regions. Preserved MFT (pMFT) was the sum of the saved MFT. The percentage of pFMT (ppMFT) was also calculated. Fascia surface (FS) was measured on MRI and it was combined with FP score resulting in preserved FS (pFS) and percentage of pFS (ppFS). FP score, pMFT, ppMFT, pFS and ppFS were significantly lower (p

Background Ewing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011. Methods The CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time. Results The hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels. Conclusions The fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3352-z) contains supplementary material, which is available to authorized users.

The use of mammalian cells for therapeutic applications is finding its way into modern medicine. However, modification or “training” of cells to make them suitable for a specific application remains complex. By envisioning a chemical toolbox that enables specific, but straight-forward and generic cellular functionalization, we investigated how membrane-receptor (pre)targeting could be combined with supramolecular host-guest interactions based on β-cyclodextrin (CD) and adamantane (Ad). The feasibility of this approach was studied in cells with membranous overexpression of the chemokine receptor 4 (CXCR4). By combining specific targeting of CXCR4, using an adamantane (Ad)-functionalized Ac-TZ14011 peptide (guest; KD = 56 nM), with multivalent host molecules that entailed fluorescent β-CD-Poly(isobutylene-alt-maleic-anhydride)-polymers with different fluorescent colors and number of functionalities, host-guest cell-surface modifications could be studied in detail. A second set of Ad-functionalized entities enabled introduction of additional surface functionalities. In addition, the attraction between CD and Ad could be used to drive cell-cell interactions. Combined we have shown that supramolecular interactions, that are based on specific targeting of an overexpressed membrane-receptor, allow specific and stable, yet reversible, surface functionalization of viable cells and how this approach can be used to influence the interaction between cells and their surroundings.

Purpose This study was designed to examine the feasibility of combining lymphoscintigraphy and intraoperative sentinel node identification in patients with head and neck melanoma by using a hybrid protein colloid that is both radioactive and fluorescent. Methods Eleven patients scheduled for sentinel node biopsy in the head and neck region were studied. Approximately 5 h before surgery, the hybrid nanocolloid labeled with indocyanine green (ICG) and technetium-99m (99mTc) was injected intradermally in four deposits around the scar of the primary melanoma excision. Subsequent lymphoscintigraphy and single photon emission computed tomography with computed tomography (SPECT/CT) were performed to identify the sentinel nodes preoperatively. In the operating room, patent blue dye was injected in 7 of the 11 patients. Intraoperatively, sentinel nodes were acoustically localized with a gamma ray detection probe and visualized by using patent blue dye and/or fluorescence-based tracing with a dedicated near-infrared light camera. A portable gamma camera was used before and after sentinel node excision to confirm excision of all sentinel nodes. Results A total of 27 sentinel nodes were preoperatively identified on the lymphoscintigraphy and SPECT/CT images. All sentinel nodes could be localized intraoperatively. In the seven patients in whom blue dye was used, 43% of the sentinel nodes stained blue, whereas all were fluorescent. The portable gamma camera identified additional sentinel nodes in two patients. Ex vivo, all radioactive lymph nodes were fluorescent and vice versa, indicating the stability of the hybrid tracer. Conclusions ICG–99mTc-nanocolloid allows for preoperative sentinel node visualization and concomitant intraoperative radio- and fluorescence guidance to the same sentinel nodes in head and neck melanoma patients. Electronic supplementary material The online version of this article (doi:10.1245/s10434-011-2180-7) contains supplementary material, which is available to authorized users.

The purpose of this study was to compare the lymphoscintigraphic drainage patterns of a hybrid sentinel node tracer consisting of the fluorescent dye indocyanine green (ICG) and 99mTc-nanocolloid with the drainage pattern of 99mTc-nanocolloid alone, the current standard tracer in many European countries. Methods: Twenty-five patients with a melanoma in the head and neck region (n = 10), a melanoma on the trunk (n = 6), or penile carcinoma (n = 9) who were scheduled for sentinel node biopsy were prospectively included. First, the standard 99mTc-nanocolloid procedure was performed. After injection at the lesion site, lymphoscintigraphy was performed with a 10-min dynamic study and static planar images at 10 min and 2 h after injection, followed by SPECT/CT. The same scintigraphic procedure was repeated after injection of hybrid ICG–99mTc-nanocolloid the same afternoon in 10 patients or the next morning in 15 patients. The paired images of both injections were evaluated, and count rates in the sentinel nodes were calculated and compared. Sentinel nodes were surgically localized using blue dye, a γ-ray detection probe, a portable γ-camera, and a fluorescence camera. Results: Lymphatic drainage was visualized in all 25 patients using 99mTc-nanocolloid, leading to the identification of 66 sentinel nodes in total. These same sentinel nodes were also identified during the second scintigraphic procedure with ICG–99mTc-nanocolloid. Moreover, a high correlation between the radioactive counting rates in the sentinel nodes of both scintigraphic studies was observed (mean R2 = 0.83). Intraoperatively (4–23 h after the second injection), all preoperatively identified sentinel nodes could be localized using radio- and fluorescence guidance combined. In total, 95% of the sentinel nodes could be intraoperatively visualized by means of fluorescence imaging, whereas merely 54% stained blue. Ex vivo, all radioactive sentinel nodes were fluorescent and vice versa. No adverse reactions were observed. Conclusion: The lymphatic drainage pattern of ICG–99mTc-nanocolloid is identical to that of 99mTc-nanocolloid. This observation, together with the added value of intraoperative fluorescence guidance, warrants wider evaluation of hybrid ICG–99mTc-nanocolloid as a tracer for sentinel node procedures.

Background Integration of molecular imaging and in particular intraoperative image guidance is expected to improve the surgical accuracy of laparoscopic lymph node (LN) dissection. Objective To show the applicability of combining preoperative, intraoperative, and postoperative sentinel node imaging using an integrated diagnostic approach based on an imaging agent that is both radioactive and fluorescent. Design, setting, and participants Before surgery, multimodal indocyanine green (ICG)- 99m Tc-NanoColl was injected into the prostate. Subsequent lymphoscintigraphy and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of pelvic nodes was performed to determine the location of the sentinel lymph nodes (SLNs) preoperatively. During the surgical procedure a fluorescence laparoscope, optimized for detection in the near infrared range, was used to visualize the nodes identified on SPECT/CT. Eleven patients scheduled for robot-assisted laparoscopic prostatectomy (RALP) with an increased risk of nodal metastasis, based on Memorial Sloan-Kettering Cancer Center/Kattan nomogram estimation, participated in a pilot assessment (N09IGF). Surgical procedure Patients underwent RALP with LN dissection for prostate cancer. Measurements Radioactive and fluorescent signals were monitored using different modalities, and the correlation between the two types of signals was studied. The location of preoperatively detected SLNs was documented. Results and limitations Preoperatively, SLNs were identified by SPECT/CT, and the multimodal nature of the imaging agent also enabled intraoperative detection via fluorescence imaging. Fluorescence particularly improved surgical guidance in areas with a high radioactive background signal such as the injection site. Ex vivo analysis revealed a strong correlation between the radioactive and fluorescent content in the excised LNs. Fluorescence detection is limited by the severe tissue attenuation of the signal. Therefore, radio guidance to the areas of interest is still desirable. Conclusions Initial data indicate that multimodal ICG- 99m Tc-NanoColloid, in combination with a laparoscopic fluorescence laparoscope, can be used to facilitate and optimize dissection of SLNs during RALP procedures.

Colon cancer prevention currently relies on colonoscopy using white light to detect and remove polyps, but small and flat polyps are difficult to detect and frequently missed when using this technique. Fluorescence colonoscopy combined with a fluorescent probe specific for a polyp biomarker may improve polyp detection. Here we describe GE-137, a water-soluble probe consisting of a 26-amino acid cyclic peptide that binds the human tyrosine kinase c-Met conjugated to a fluorescent cyanine dye. Intravenous administration of GE-137 leads to its accumulation specifically in c-Met-expressing tumors in mice, and it is safe and well tolerated in humans. Fluorescence colonoscopy in patients receiving intravenous GE-137 enabled visualization of all neoplastic polyps that were visible with white light (38), as well as an additional nine polyps that were not visible with white light. This first-in-human pilot study shows that molecular imaging using an intravenous fluorescent agent specific for c-Met is feasible and safe, and that it may enable the detection of polyps missed by other techniques.

In trauma and orthopedic surgery, infection of implants has a major impact on the outcome for patients. Infections may develop either during the initial implantation or during the lifetime of an implant. Both infections, as well as aseptic loosening of the implant, are reasons for revision of the implants. Therefore, discrimination between aseptic-mechanical-loosening and septic-bacterial-loosening of implants is critical during selection of a patient-tailored treatment policy. Specific detection and visualization of infections is a challenge because it is difficult to discriminate infections from inflammation. An imaging tracer that facilitates bacterial identification in a pre- and intraoperative setting may aid the workup for patients suspicious of bacterial infections. In this study we evaluated an antimicrobial peptide conjugated to a hybrid label, which contains both a radioisotope and a fluorescent dye. After synthesis of DTPA-Cy5-UBI29-41 and-when necessary-radiolabeling with (111)In (yield 96.3 ± 2.7%), in vitro binding to various bacterial strains was evaluated using radioactivity counting and confocal fluorescence microscopy. Intramuscular bacterial infections (S. aureus or K. pneumoniae) were also visualized in vivo using a combined nuclear and fluorescence imaging system. The indium-111 was chosen as label as it has a well-defined coordination chemistry, and in pilot studies labeling DTPA-Cy5-UBI29-41 with technetium-99m, we encountered damage to the Cy5 dye after the reduction with SnCl2. As a reference, we used the validated tracer (99m)Tc-UBI29-41. Fast renal excretion of (111)In-DTPA-Cy5-UBI29-41 was observed. Target to nontarget (T/NT) ratios were highest at 2 h post injection: radioactivity counting yielded T/NT ratios of 2.82 ± 0.32 for S. aureus and 2.37 ± 0.05 for K. pneumoniae. Comparable T/NT ratios with fluorescence imaging of 2.38 ± 0.09 for S. aureus and 3.55 ± 0.31 for K. pneumoniae were calculated. Ex vivo confocal microscopy of excised infected tissues showed specific binding of the tracer to bacteria. Using a combination of nuclear and fluorescence imaging techniques, the hybrid antimicrobial peptide conjugate DTPA-Cy5-UBI29-41 was shown to specifically accumulate in bacterial infections. This hybrid tracer may facilitate integration of noninvasive identification of infections and their extent as well as real-time fluorescence guidance during surgical resection of infected areas.

UnlabelledScreening of biomarker expression levels in tumor biopsy samples not only provides an assessment of prognostic and predictive factors, but may also be used for selection of biomarker-specific imaging strategies. To assess the feasibility of using a biopsy specimen for a personalized selection of an imaging agent, the chemokine receptor 4 (CXCR4) was used as a reference biomarker.MethodsA hybrid CXCR4 targeting peptide (MSAP-Ac-TZ14011) containing a fluorescent dye and a chelate for radioactive labeling was used to directly compare initial flow cytometry-based target validation in fresh tumor tissue to in vivo single photon emission computed tomography (SPECT) imaging and in vivo and ex vivo fluorescence imaging.ResultsFlow cytometric analysis of mouse tumor derived cell suspensions enabled discrimination between 4T1 control tumor lesions (with low levels of CXCR4 expression) and CXCR4 positive early, intermediate and late stage MIN-O lesions based on their CXCR4 expression levels; CXCR4(basal), CXCR4(+) and CXCR4(++) cell populations could be accurately discriminated. Mean fluorescent intensity ratios between expression in MIN-O and 4T1 tissue found with flow cytometry were comparable to ratios obtained with in vivo SPECT/CT and fluorescence imaging, ex vivo fluorescence evaluation and standard immunohistochemistry.ConclusionThe hybrid nature of a targeting imaging agent like MSAP-Ac-TZ14011 enables integration of target selection, in vivo imaging and ex vivo validation using a single agent. The use of biopsy tissue for biomarker screening can readily be expanded to other targeting hybrid imaging agents and can possibly help increase the clinical applicability of tumor-specific imaging approaches.

Intraprostatic injection of the hybrid tracer indocyanine green (ICG)-(99m)Tc-nanocolloid enables both preoperative sentinel node (SN) identification and intraoperative visualization of the SN. Relating the fluorescence deposits in embedded prostate tissue specimens to the preoperatively detected SNs also provides the opportunity to study the influence of their placement on lymphatic drainage pattern.Nineteen patients with prostate carcinoma scheduled for robot-assisted laparoscopic prostatectomy and lymph node (LN) dissection were included. ICG-(99m)Tc-nanocolloid was injected intraprostatically, guided by ultrasound. SN biopsy was performed using a combination of radioguidance and fluorescence guidance. Tracer distribution was visualized in paraffin-embedded prostate samples using ex vivo fluorescence imaging. This distribution was correlated to the number and location of the SNs identified on preoperative lymphoscintigraphy and SPECT/CT.ICG-(99m)Tc-nanocolloid helped guide surgical excision of the SNs. Ex vivo fluorescence imaging revealed a large variation in the locations of intraprostatic tracer deposits among patients. Tracer deposits in the peripheral zone correlated with a higher number of visualized LNs than deposits in the central zone (on average, 4.7 vs. 2.4 LNs per patient). Furthermore, tracer deposits in the mid gland correlated with a higher number of visualized LNs than deposits near the base or apex of the prostate (on average, 6 vs. 3.5 LNs per patient).The hybrid nature of the tracer not only enables surgical guidance but also provides an opportunity to study the correlation between the location of tracer deposits within the prostate and the number and location of preoperatively visualized SNs. These data suggest that the location at which a tracer deposit is placed influences the lymphatic drainage pattern.

Hybrid tracers that are both radioactive and fluorescent help extend the use of fluorescence-guided surgery to deeper structures. Such hybrid tracers facilitate preoperative surgical planning using (3D) scintigraphic images and enable synchronous intraoperative radio- and fluorescence guidance. Nevertheless, we previously found that improved orientation during laparoscopic surgery remains desirable. Here we illustrate how intraoperative navigation based on optical tracking of a fluorescence endoscope may help further improve the accuracy of hybrid surgical guidance. After feeding SPECT/CT images with an optical fiducial as a reference target to the navigation system, optical tracking could be used to position the tip of the fluorescence endoscope relative to the preoperative 3D imaging data. This hybrid navigation approach allowed us to accurately identify marker seeds in a phantom setup. The multispectral nature of the fluorescence endoscope enabled stepwise visualization of the two clinically approved fluorescent dyes, fluorescein and indocyanine green. In addition, the approach was used to navigate toward the prostate in a patient undergoing robot-assisted prostatectomy. Navigation of the tracked fluorescence endoscope toward the target identified on SPECT/CT resulted in real-time gradual visualization of the fluorescent signal in the prostate, thus providing an intraoperative confirmation of the navigation accuracy.

Human serum albumin (HSA) is a biological nanocarrier that forms non-covalent complexes with a number of synthetic and biomolecules. Previously we demonstrated radiolabeled HSA-based nanoparticles can form non-covalent complexes with fluorescent cyanine dyes yielding imaging agents for surgical guidance towards tumor draining lymph nodes. Here the self-assembly approach enabled rapid clinical translation. Based on this experience we reasoned it would be interesting to expand this non-covalent technology to a targeted approach. Therefore, the ability of HSA to form non-covalent self-assembled complexes with peptides via near-infrared (NIR) cyanine dyes was explored. Foster resonance energy transfer (FRET) quenching interactions between HSA-Cy5 and the non-covalently bound fluorescent molecules indocyanine green (ICG), IR783-CO(2)H and three IR783-labeled targeting peptides were used to monitor complex assembly and disassembly. The host-guest interactions between HSA and IR783-labeled peptides enabled the formation of (bio)nanoparticles that are coated with peptides, which may target α(v)β(3)-integrins, the chemokine receptor 4 (CXCR4), or somatostatin receptors. The potential of CXCR4-targeted (bio)nanoparticles in sentinel lymph node procedures is demonstrated in vivo. By non-covalently binding NIR-dye labeled peptides to an already clinically approved HSA-scaffold, we have readily formed targeted bionanoparticles.

One clinical precedent for the use of nanosized imaging agents is the localization of the tumor draining sentinel lymph nodes. In this application, radiocolloids such as (99m)Tc-NanoColl are currently used to plan the surgical procedure and to provide acoustic guidance during the intervention. Additional injections of dyes are common to provide optical surgical guidance. Bimodal imaging agents, which are both radioactive and fluorescent, have the potential to be used for both surgical planning and intraoperative fluorescence guidance towards the sentinel lymph nodes. This review provides an overview of the radioactive, fluorescent, and size properties of (non-targeted) bimodal nanoparticles, and their (potential) value in sentinel lymph node detection.

Fluorescence molecular imaging is rapidly increasing its popularity in image guided surgery applications. To help develop its full surgical potential it remains a challenge to generate dual-emissive imaging agents that allow for combined visible assessment and sensitive camera based imaging. To this end, we now describe multispectral InP/ZnS quantum dots (QDs) that exhibit a bright visible green/yellow exciton emission combined with a long-lived far red defect emission. The intensity of the latter emission was enhanced by X-ray irradiation and allows for: 1) inverted QD density dependent defect emission intensity, showing improved efficacies at lower QD densities, and 2) detection without direct illumination and interference from autofluorescence.

Using a "spontaneous" mammary mouse tumor model we set out to develop diagnostic approaches for non-invasive P-glycoprotein (P-gp) staging and response prediction.(99m)Tc-MIBI efflux rates were measured using a gamma camera in three Brca1 (-/-); p53 (-/-) mouse mammary tumors that have different Mdr1a/b expression levels. The efflux rates were quantified in the 10-30-min period after injection. In addition to the P-gp-mediated efflux measurements in untreated tumors, efflux measurements were performed in the presence of the P-gp inhibitor tariquidar. Volumetric doxorubicin response patterns for the different tumors were determined and correlated with the efflux rates.Combined pre- and post-inhibitor treatment imaging of P-gp-mediated efflux correlated with Mdr1a/b expression: basal (0.0026, p = 0.16), 3-fold Mdr1a/b (0.0074, p = 0.02), and 17-fold Mdr1a and 46-fold Mdr1b (0.012, p = 0.002). Based on the doxorubicin response of these tumors, we generated a computer-aided diagnosis model that predicts the likelihood of drug resistance.Quantified (99m)Tc-MIBI efflux has potential to: (1) noninvasively assign Mdr1 expression levels, (2) predict the therapeutic impact of a P-gp inhibitor, and (3) noninvasively assess the probability of drug resistance.

A sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) assay has been developed and validated for quantification of total topotecan in human and mouse plasma and in mouse tissue samples. Isocratic separation was achieved on a Zorbax SB-C(18) column and topotecan was monitored fluorimetrically. Two ranges of calibrations curves were used to determine lower levels of topotecan more accurately. Acceptable accuracy and precision was achieved for all matrices. Topotecan was stable upon repeated freeze-thawing for three cycles or storage for 24 h at ambient temperatures in spiked plasma samples and tissue homogenates, except in heart homogenates. In an additional validation experiment in which (14)C-labeled topotecan was administered to mice, the levels of unchanged topotecan were about 80-90% of the total radioactivity in tissue homogenates collected 10 min after drug administration and virtually similar as in plasma samples. However, results in tissue homogenates obtained 4 h post-drug administration indicated substantial metabolism of topotecan. This assay is suitable for studying the pharmacokinetics and tissue distribution of topotecan in mice. Our results demonstrate the importance of including all tissues of interest for pharmacokinetic studies in the validation procedure.

A relatively new strategy to longitudinally monitor tumor load in intact animals and the effects of therapy is noninvasive bioluminescence imaging (BLI). The validity of BLI for quantitative assessment of tumor load in small animals is critically evaluated in the present review. Cancer cells are grafted in mice or rats after transfection with a luciferase gene—usually that of a firefly. To determine tumor load, animals receive the substrate agent luciferin intraperitoneally, which luciferase converts into oxyluciferin in an ATP-dependent manner. Light emitted by oxyluciferin in viable cancer cells is captured noninvasively with a highly sensitive charge-coupled device (CCD) camera. Validation studies indicate that BLI is useful to determine tumor load in the course of time, with each animal serving as its own reference. BLI is rapid, easy to perform, and sensitive. It can detect tumor load shortly after inoculation, even when relatively few cancer cells (2500–10,000) are used. BLI is less suited for the determination of absolute tumor mass in an animal because of quenching of bioluminescence by tissue components and the exact location of tumors because its spatial resolution is limited. Nevertheless, BLI is a powerful tool for high-throughput longitudinal monitoring of tumor load in small animals and allows the implementation of more advanced orthotopic tumor models in therapy intervention studies with almost the same simplicity as when measuring traditional ectopic subcutaneous models in combination with calipers.

Trabectedin (ET-743, Yondelis™) is a novel anticancer drug currently undergoing phase II and III investigations. There are various and conflicting reports whether trabectedin is a substrate for P-glycoprotein (P-gp), an important factor in drug disposition and multi-drug resistance (MDR). We have now unambiguously shown that trabectedin is a P-gp substrate by investigating vectorial transport over monolayers of LLC-PK1 pig kidney and Madine-Darby Canine kidney (MDCK) cells and the mdr1a and/or MDR1 transfected subclones. We further characterized the cytotoxic effects and cellular accumulation of trabectedin in these cell lines as well as in a panel of other cell lines with high or moderate expression levels of P-gp. Trabectedin displayed the typical MDR phenotype only in highly P-gp expressing cell lines, but not in cell lines with expression levels more closely conforming to clinical samples, suggesting that P-gp will not confer resistance to trabectedin in cancer patients.

P-glycoprotein seems to be the most important factor limiting the oral absorption of paclitaxel. We have now explored the mechanisms responsible for the low oral bioavailability of docetaxel, a structurally related taxane drug. The recovery of 33% of oxidative metabolites and only 39% of unchanged drug in the feces of FVB wild-type mice receiving 10 mg/kg of oral docetaxel indicates that the major part of the oral dose has been absorbed. The feces and bile of mice receiving 10 mg/kg of i.v. docetaxel contained large amounts of metabolites and only minor quantities of unchanged drug, highlighting the importance of metabolism as an elimination route for this drug. In wild-type and P-glycoprotein knockout mice, dose escalation of p.o. administered docetaxel from 10 to 30 mg/kg resulted in a more than proportional increase in plasma levels, which suggested saturation of first-pass metabolism. Moreover, coadministration of 12.5 mg/kg of the HIV protease inhibitor ritonavir, also a strong inhibitor of cytochrome P4503A4 with only minor P-glycoprotein inhibiting properties, increased the plasma levels after oral docetaxel by 50-fold. In vitro transport studies across monolayers of LLC-PK1 cells (parental and transduced with MDR1 or Mdr1a) suggested that docetaxel is a weaker substrate for P-glycoprotein than paclitaxel is. In conclusion, docetaxel is well absorbed from the gut lumen in mice despite the presence of P-glycoprotein in the gut wall. Subsequent first-pass extraction is the most important factor determining its low bioavailability. The inhibition of docetaxel metabolism by ritonavir provides an interesting strategy to improve the systemic exposure of oral docetaxel.

Accurate tumor excision is crucial in the locoregional treatment of cancer, and for this purpose, surgeons often rely on guide wires or radioactive markers for guidance toward the lesion. Further improvement may be obtained by adding optical guidance to currently used methods, in the form of intra-operative fluorescence imaging. To achieve such a multimodal approach, we have generated markers that can be used in a pre-, intra-, and post-operative setting, based on a cocktail of a dual-emissive inorganic dye, lipids, and pertechnetate. Phantom experiments demonstrate that these seeds can be placed accurately around a surrogate tumor using ultrasound. Three-dimensional bracketing provides delineation of the entire lesion. Combined with the multimodal nature, this provides the opportunity to predetermine the resection margins by validating the placement accuracy using multiple imaging modalities (namely, x ray, MRI, SPECT/CT, and ultrasound). The dual-emissive fluorescent properties of the dye provide the unique opportunity to intra-operatively estimate the depth of the seed in the tissue via multispectral imaging: emission green λmax=520 nm≤5 mm penetration versus emission red λmax=660 nm≤12 mm penetration. By using particles with different colors, the original geographic orientation of the excised tissue can be determined.