Identification of impactful imaging biomarker: Clinical applications for breast and prostate carcinoma. / Ermittlung aussagekräftiger Bildgebungsbiomarker: Klinische Anwendungen bei Mamma- und Prostatakarzinomen.

BACKGROUND: Imaging biomarkers are quantitative parameters from imaging modalities, which are collected noninvasively, allow conclusions about physiological and pathophysiological processes, and may consist of single (monoparametric) or multiple parameters (bi- or multiparametric). METHOD: This review aims to present the state of the art for the quantification of multimodal and multiparametric imaging biomarkers. Here, the use of biomarkers using artificial intelligence will be addressed and the clinical application of imaging biomarkers in breast and prostate cancers will be explained. For the preparation of the review article, an extensive literature search was performed based on Pubmed, Web of Science and Google Scholar. The results were evaluated and discussed for consistency and generality. RESULTS AND CONCLUSION: Different imaging biomarkers (multiparametric) are quantified based on the use of complementary imaging modalities (multimodal) from radiology, nuclear medicine, or hybrid imaging. From these techniques, parameters are determined at the morphological (e. g., size), functional (e. g., vascularization or diffusion), metabolic (e. g., glucose metabolism), or molecular (e. g., expression of prostate specific membrane antigen, PSMA) level. The integration and weighting of imaging biomarkers are increasingly being performed with artificial intelligence, using machine learning algorithms. In this way, the clinical application of imaging biomarkers is increasing, as illustrated by the diagnosis of breast and prostate cancers. KEY POINTS: · Imaging biomarkers are quantitative parameters to detect physiological and pathophysiological processes.. · Imaging biomarkers from multimodality and multiparametric imaging are integrated using artificial intelligence algorithms.. · Quantitative imaging parameters are a fundamental component of diagnostics for all tumor entities, such as for mammary and prostate carcinomas.. CITATION FORMAT: · Bäuerle T, Dietzel M, Pinker K et al. Identification of impactful imaging biomarker: Clinical applications for breast and prostate carcinoma. Fortschr Röntgenstr 2024; 196: 354 - 362.

Intravenous Injection of PEI-Decorated Iron Oxide Nanoparticles Impacts NF-kappaB Protein Expression in Immunologically Stressed Mice.

Nanoparticle-based formulations are considered valuable tools for diagnostic and treatment purposes. The surface decoration of nanoparticles with polyethyleneimine (PEI) is often used to enhance their targeting and functional properties. Here, we aimed at addressing the long-term fate in vivo and the potential "off-target" effects of PEI decorated iron oxide nanoparticles (PEI-MNPs) in individuals with low-grade and persistent systemic inflammation. For this purpose, we synthesized PEI-MNPs (core-shell method, PEI coating under high pressure homogenization). Further on, we induced a low-grade and persistent inflammation in mice through regular subcutaneous injection of pathogen-associated molecular patterns (PAMPs, from zymosan). PEI-MNPs were injected intravenously. Up to 7 weeks thereafter, the blood parameters were determined via automated fluorescence flow cytometry, animals were euthanized, and the organs analyzed for iron contents (atomic absorption spectrometry) and for expression of NF-κB associated proteins (p65, IκBα, p105/50, p100/52, COX-2, Bcl-2, SDS-PAGE and Western blotting). We observed that the PEI-MNPs had a diameter of 136 nm and a zeta-potential 56.9 mV. After injection in mice, the blood parameters were modified and the iron levels were increased in different organs. Moreover, the liver of animals showed an increased protein expression of canonical NF-κB signaling pathway members early after PEI-MNP application, whereas at the later post-observation time, members of the non-canonical signaling pathway were prominent. We conclude that the synergistic effect between PEI-MNPs and the low-grade and persistent inflammatory state is mainly due to the hepatocytes sensing infection (PAMPs), to immune responses resulting from the intracellular metabolism of the uptaken PEI-MNPs, or to hepatocyte and immune cell communications. Therefore, we suggest a careful assessment of the safety and toxicity of PEI-MNP-based carriers for gene therapy, chemotherapy, and other medical applications not only in healthy individuals but also in those suffering from chronic inflammation.

Hyperthermia Influences the Secretion Signature of Tumor Cells and Affects Endothelial Cell Sprouting.

Tumors are a highly heterogeneous mass of tissue showing distinct therapy responses. In particular, the therapeutic outcome of tumor hyperthermia treatments has been inconsistent, presumably due to tumor versus endothelial cell cross-talks related to the treatment temperature and the tumor tissue environment. Here, we investigated the impact of the average or strong hyperthermic treatment (43 °C or 47 °C for 1 h) of the human pancreatic adenocarcinoma cell line (PANC-1 and BxPC-3) on endothelial cells (HUVECs) under post-treatment normoxic or hypoxic conditions. Immediately after the hyperthermia treatment, the distinct repression of secreted pro-angiogenic factors (e.g., VEGF, PDGF-AA, PDGF-BB, M-CSF), intracellular HIF-1α and the enhanced phosphorylation of ERK1/2 in tumor cells were detectable (particularly for strong hyperthermia, 2D cell monolayers). Notably, there was a significant increase in endothelial sprouting when 3D self-organized pancreatic cancer cells were treated with strong hyperthermia and the post-treatment conditions were hypoxic. Interestingly, for the used treatment temperatures, the intracellular HIF-1α accumulation in tumor cells seems to play a role in MAPK/ERK activation and mediator secretion (e.g., VEGF, PDGF-AA, Angiopoietin-2), as shown by inhibition experiments. Taken together, the hyperthermia of pancreatic adenocarcinoma cells in vitro impacts endothelial cells under defined environmental conditions (cell-to-cell contact, oxygen status, treatment temperature), whereby HIF-1α and VEGF secretion play a role in a complex context. Our observations could be exploited for the hyperthermic treatment of pancreatic cancer in the future.

Local Magnetic Hyperthermia and Systemic Gemcitabine/Paclitaxel Chemotherapy Triggers Neo-Angiogenesis in Orthotopic Pancreatic Tumors without Involvement of Auto/Paracrine Tumor Cell VEGF Signaling and Hypoxia.

There is a growing interest in exploring the therapeutically mediated modulation of tumor vascularization of pancreatic cancer, which is known for its poorly perfused tumor microenvironment limiting the delivery of therapeutic agents to the tumor site. Here, we assessed how magnetic hyperthermia in combination with chemotherapy selectively affects growth, the vascular compartment of tumors, and the presence of tumor cells expressing key regulators of angiogenesis. To that purpose, a orthotopic PANC-1 (fluorescent human pancreatic adenocarcinoma) mouse tumor model (Rj:Athym-Foxn1nu/nu) was used. Magnetic hyperthermia was applied alone or in combination with systemic chemotherapy (gemcitabine 50 mg per kg body weight, nab-pacitaxel 30 mg/kg body weight) on days 1 and 7 following magnetic nanoparticle application (dose: 1 mg per 100 mm3 of tumor). We used ultrasound imaging, immunohistochemistry, multi-spectral optoacoustic tomography (MSOT), and hematology to assess the biological parameters mentioned above. We found that magnetic hyperthermia in combination with gemcitabine/paclitaxel chemotherapy was able to impact tumor growth (decreased volumes and Ki67 expression) and to trigger neo-angiogenesis (increased small vessel diameter) as a result of the therapeutically mediated cell damages/stress in tumors. The applied stressors activated specific pro-angiogenic mechanisms, which differed from those seen in hypoxic conditions involving HIF-1α, since (a) treated tumors showed a significant decrease of cells expressing VEGF, CD31, HIF-1α, and neuropilin-1; and (b) the relative tumor blood volume and oxygen level remained unchanged. Neo-angiogenesis seems to be the result of the activation of cell stress pathways, like MAPK pathways (high number of pERK-expressing tumor cells). In the long term, the combination of magnetic hyperthermia and chemotherapy could potentially be applied to transiently modulate tumor angiogenesis and to improve drug accessibility during oncologic therapies of pancreatic cancer.

Silencing of proinflammatory NF-κB and inhibition of herpes simplex virus (HSV) replication by ultrasmall gold nanoparticles (2 nm) conjugated with small-interfering RNA.

Azide-terminated ultrasmall gold nanoparticles (2 nm gold core) were covalently functionalized with alkyne-terminated small-interfering siRNA duplexes by copper-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry). The nanoparticle core was visualized by transmission electron microscopy. The number of attached siRNA molecules per nanoparticle was determined by a combination of atomic absorption spectroscopy (AAS; for gold) and UV-Vis spectroscopy (for siRNA). Each nanoparticle carried between 6 and 10 siRNA duplex molecules which corresponds to a weight ratio of siRNA to gold of about 2.2 : 1. Different kinds of siRNA were conjugated to the nanoparticles, depending on the gene to be silenced. In general, the nanoparticles were readily taken up by cells and highly efficient in gene silencing, in contrast to free siRNA. This was demonstrated in HeLa-eGFP cells (silencing of eGFP) and in LPS-stimulated macrophages (silencing of NF-κB). Furthermore, we demonstrated that nanoparticles carrying antiviral siRNA potently inhibited the replication of Herpes simplex virus 2 (HSV-2) in vitro. This highlights the strong potential of siRNA-functionalized ultrasmall gold nanoparticles in a broad spectrum of applications, including gene silencing and treatment of viral infections, combined with a minimal dose of gold.

[The world of light in diagnostic medicine : Current developments in optical imaging]. / Lichtwelten in der diagnostischen Medizin : Aktuelle Entwicklungen in der optischen Bildgebung.

Optical imaging has always played a central role in the elucidation of biological and physiological mechanisms in modern biology and medicine. Based on the good experiences in light microscopy, sophisticated meso- and macroscopic optical imaging systems have recently been created. All optical imaging methods are characterized by high user-friendliness and sensitivity, they are associated with relatively low costs and do not require any radioactivity. Its clinical performance is seen in the intraoperative imaging of the tissue area to be removed and in the radiotracer-free diagnosis of diseases in body areas with good accessibility to light. From a pathomorphological point of view, the focus was particularly on the depiction of tumors and inflammation. Imaging detection of fluorescent dyes with emission characteristics in the near-infrared range of the spectrum is favorable in terms of a good signal-to-background ratio and improved information acquisition from greater tissue depths. A major challenge, however, is the diverse photon interactions with the tissue. Previous research and development work has produced various in vivo optical imaging methods, some of which are still in the experimental stage (e.g., fluorescence-mediated tomography, multispectral in vivo imaging, bioluminescence imaging, Raman spectroscopy), while others already have made their way into the clinical setting (e.g., fluorescence reflection imaging, optoacoustic imaging). The most important optical methods are presented in this review article.

The Peptide/Antibody-Based Surface Decoration of Calcium Phosphate Nanoparticles Carrying siRNA Influences the p65 NF-κB Protein Expression in Inflamed Cells In Vitro.

Earlier studies with nanoparticles carrying siRNA were restricted to investigating the inhibition of target-specific protein expression, while almost ignoring effects related to the nanoparticle composition. Here, we demonstrate how the design and surface decoration of nanoparticles impact the p65 nuclear factor-kappa B (NF-κB) protein expression in inflamed leucocytes and endothelial cells in vitro. We prepared silica-coated calcium phosphate nanoparticles carrying encapsulated siRNA against p65 NF-κB and surface-decorated with peptides or antibodies. We show that RGD-decorated nanoparticles are efficient in down-regulating p65 NF-κB protein expression in endothelial cells as a result of an enhanced specific cellular binding and subsequent uptake of nanoparticles. In contrast, nanoparticles decorated with IgG (whether specific or not for CD69) are efficient in down-regulating p65 NF-κB protein expression in T-cells, but not in B-cells. Thus, an optimized nanoparticle decoration with xenogenic IgG may stimulate a specific cellular uptake. In summary, the composition of siRNA-loaded calcium phosphate nanoparticles can either weaken or stimulate p65 NF-κB protein expression in targeted inflamed leucocytes and endothelial cells. In general, unveiling such interactions may be very useful for the future design of anti-p65 siRNA-based nanomedicines for treatment of inflammation-associated diseases.

Drug loss from Paclitaxel-Coated Balloons During Preparation, Insertion and Inflation for Angioplasty: A Laboratory Investigation.

PURPOSE: To investigate drug contamination of the working environment with paclitaxel drug-coated balloon (DCB) angioplasty due to loss of paclitaxel containing particles from the coating during DCB preparation, insertion, and inflation. MATERIAL AND METHODS: In an experimetal laboratory setting, drug loss during removal of the protective cover and insertion of the DCB through the hemostatic valve of the introducer sheath and after inflation was examined. In seven DCB types of different manufacturers, semi-quantitative image analysis was performed during five standardized tests cycles. Additionally, every DCB type passed one cycle of a wipe test and one cycle of air sampling. RESULTS: By removing the protective cover, the paclitaxel-covered balloon surface was significantly reduced in 3 out of 7 products (P = 0.043). Overall, extend of decline ranged from 0.4 to 12%. In 6 of 7 products, powdered paclitaxel clusters dropped down upon removal of the protective cover (0.099 ng/cm2 up to approx. 22 ng/cm2). Contamination of the air was detected in none of the DCB types. When pushed through the vascular sheath, none of the investigated DCB types showed a significant loss of paclitaxel from the coated balloon surface. After balloon inflation, the paclitaxel-coated surface area varied between manufacturers ranging from 25.9 to 97.8%. CONCLUSION: In some DCB types, the removal of the protective cover already leads to a significant loss of paclitaxel and paclitaxel-coated surfaces. As a result, there will be a contamination of the workplace and a reduction in the therapeutic dose. LEVEL OF EVIDENCE: No level of evidence.

Nanoparticles Carrying NF-κB p65-Specific siRNA Alleviate Colitis in Mice by Attenuating NF-κB-Related Protein Expression and Pro-Inflammatory Cellular Mediator Secretion.

Ulcerative colitis is a disease that causes inflammation and ulcers in the colon and which is typically recurrent, and NF-κB proteins are important players during disease progression. Here, we assess the impact of silica-coated calcium phosphate nanoparticles carrying encapsulated siRNA against NF-κB p65 on a murine model of colitis. To this end, nanoparticles were injected intravenously (2.0 mg siRNA/kg body weight) into mice after colitis induction with dextran sulfate sodium or healthy ones. The disease activity index, the histopathological impact on the colon, the protein expression of several NF-κB-associated players, and the mediator secretion (colon tissue, blood) were analyzed. We found that the nanoparticles effectively alleviated the clinical and histopathological features of colitis. They further suppressed the expression of NF-κB proteins (e.g., p65, p50, p52, p100, etc.) in the colon. They finally attenuated the local (colon) or systemic (blood) pro-inflammatory mediator secretion (e.g., TNF-α, IFN-ß, MCP-1, interleukins, etc.) as well as the leucocyte load of the spleen and mesenteric lymph nodes. The nanoparticle biodistribution in diseased animals was seen to pinpoint organs containing lymphoid entities (appendix, intestine, lung, etc.). Taken together, the nanoparticle-related silencing of p65 NF-κB protein expression could well be used for the treatment of ulcerative colitis in the future.

Iron Oxide Nanoparticles Carrying 5-Fluorouracil in Combination with Magnetic Hyperthermia Induce Thrombogenic Collagen Fibers, Cellular Stress, and Immune Responses in Heterotopic Human Colon Cancer in Mice.

In this study we looked for the main protein pathway regulators which were responsible for the therapeutic impact on colon cancers when combining magnetic hyperthermia with the chemotherapeutic agent 5-fluorouracil (5FU). To this end, chitosan-coated magnetic nanoparticles (MNP) functionalized with 5FU were intratumorally injected into subcutaneous human colon cancer xenografts (HT-29) in mice and exposed to an alternating magnetic field. A decreased tumor growth was found particularly for the combined thermo-chemotherapy vs. the corresponding monotherapies. By using computational analysis of the tumor proteome, we found upregulated functional pathway categories termed "cellular stress and injury", "intracellular second messenger and nuclear receptor signaling", "immune responses", and "growth proliferation and development". We predict TGF-beta, and other mediators, as important upstream regulators. In conclusion, our findings show that the combined thermo-chemotherapy induces thrombogenic collagen fibers which are able to impair tumor nutrient supply. Further on, we associate several responses to the recognition of damage associated molecular patterns (DAMPs) by phagocytic cells, which immigrate into the tumor area. The activation of some pathways associated with cell survival implies the necessity to conduct multiple therapy sessions in connection with a corresponding monitoring, which could possibly be performed on the base of the identified protein regulators.

MNP-Enhanced Microwave Medical Imaging by Means of Pseudo-Noise Sensing.

Magnetic nanoparticles have been investigated for microwave imaging over the last decade. The use of functionalized magnetic nanoparticles, which are able to accumulate selectively within tumorous tissue, can increase the diagnostic reliability. This paper deals with the detecting and imaging of magnetic nanoparticles by means of ultra-wideband microwave sensing via pseudo-noise technology. The investigations were based on phantom measurements. In the first experiment, we analyzed the detectability of magnetic nanoparticles depending on the magnetic field intensity of the polarizing magnetic field, as well as the viscosity of the target and the surrounding medium in which the particles were embedded, respectively. The results show a nonlinear behavior of the magnetic nanoparticle response depending on the magnetic field intensity for magnetic nanoparticles diluted in distilled water and for magnetic nanoparticles embedded in a solid medium. Furthermore, the maximum amplitude of the magnetic nanoparticles responses varies for the different surrounding materials of the magnetic nanoparticles. In the second experiment, we investigated the influence of the target position on the three-dimensional imaging of the magnetic nanoparticles in a realistic measurement setup for breast cancer imaging. The results show that the magnetic nanoparticles can be detected successfully. However, the intensity of the particles in the image depends on its position due to the path-dependent attenuation, the inhomogeneous microwave illumination of the breast, and the inhomogeneity of the magnetic field. Regarding the last point, we present an approach to compensate for the inhomogeneity of the magnetic field by computing a position-dependent correction factor based on the measured magnetic field intensity and the magnetic susceptibility of the magnetic particles. Moreover, the results indicate an influence of the polarizing magnetic field on the measured ultra-wideband signals even without magnetic nanoparticles. Such a disturbing influence of the polarizing magnetic field on the measurements should be reduced for a robust magnetic nanoparticles detection. Therefore, we analyzed the two-state (ON/OFF) and the sinusoidal modulation of the external magnetic field concerning the detectability of the magnetic nanoparticles with respect to these spurious effects, as well as their practical application.

Silica-coated calcium phosphate nanoparticles for gene silencing of NF-κB p65 by siRNA and their impact on cellular players of inflammation.

The transcription factor NF-κB and its signaling cascade both play key roles in all inflammatory processes. The most critical member of the NF-κB transcription factor family is p65. We investigated the role of cationic silica-coated calcium phosphate nanoparticles (spherical, diameter by SEM 50-60 nm; zeta potential about +26 mV; stabilized by polyethyleneimine) carrying encapsulated siRNA against NF-κB p65 and their influence on inflamed cells. The nanoparticles were taken up by cells of the blood compartment involved in the inflammatory response, particularly by monocytes, and to a lesser extent by endothelial cells and B-cells, but not by T-cells. The particles were found in endolysosomes where they were dissolved at low pH and released the siRNA into the cytoplasm. This was confirmed by dissolution experiments of model nanoparticles in simulated endolysosomal medium (pH 4.7) and by intracellular co-localization studies of double-labeled nanoparticles (using a negatively charged model peptide for siRNA). The encapsulated functional siRNA reverted the p65 gene and protein expression in inflamed monocytes, the main cells in immune response and surveillance, almost back to the non-inflammatory condition. Additionally, the nanoparticles suppressed the pro-inflammatory cytokine expression profiles (TNF-α, IL-6, IFN-ß) in inflamed J774A.1 monocytes. Taken together, such nanoparticles can be applied for the treatment of inflammatory diseases.

Deep-tissue localization of magnetic field hyperthermia using pulse sequencing.

OBJECTIVE: Deep-tissue localization of thermal doses is a long-standing challenge in magnetic field hyperthermia (MFH), and remains a limitation of the clinical application of MFH to date. Here, we show that pulse sequencing of MFH leads to a more persistent inhibition of tumor growth and less systemic impact than continuous MFH, even when delivering the same thermal dose. METHODS: We used an in vivo orthotopic murine model of pancreatic PANC-1 cancer, which was designed with a view to the forthcoming 'NoCanTher' clinical study, and featured MFH alongside systemic chemotherapy (SyC: gemcitabine and nab-paclitaxel). In parallel, in silico thermal modelling was implemented. RESULTS: Tumor volumes 27 days after the start of MFH/SyC treatment were 53% (of the initial volume) in the pulse MFH group, compared to 136% in the continuous MFH group, and 337% in the non-treated controls. Systemically, pulse MFH led to ca. 50% less core-temperature increase in the mice for a given injected dose of magnetic heating agent, and inflicted lower levels of the stress marker, as seen in the blood-borne neutrophil-to-lymphocyte ratio (1.7, compared to 3.2 for continuous MFH + SyC, and 1.2 for controls). CONCLUSION: Our data provided insights into the influence of pulse sequencing on the observed biological outcomes, and validated the nature of the improved thermal dose localization, alongside significant lowering of the overall energy expenditure entailed in the treatment.

Effect of Matrix-Modulating Enzymes on The Cellular Uptake of Magnetic Nanoparticles and on Magnetic Hyperthermia Treatment of Pancreatic Cancer Models In Vivo.

Magnetic hyperthermia can cause localized thermal eradication of several solid cancers. However, a localized and homogenous deposition of high concentrations of magnetic nanomaterials into the tumor stroma and tumor cells is mostly required. Poorly responsive cancers such as the pancreatic adenocarcinomas are hallmarked by a rigid stroma and poor perfusion to therapeutics and nanomaterials. Hence, approaches that enhance the infiltration of magnetic nanofluids into the tumor stroma convey potentials to improve thermal tumor therapy. We studied the influence of the matrix-modulating enzymes hyaluronidase and collagenase on the uptake of magnetic nanoparticles by pancreatic cancer cells and 3D spheroids thereof, and the overall impact on magnetic heating and cell death. Furthermore, we validated the effect of hyaluronidase on magnetic hyperthermia treatment of heterotopic pancreatic cancer models in mice. Treatment of cultured cells with the enzymes caused higher uptake of magnetic nanoparticles (MNP) as compared to nontreated cells. For example, hyaluronidase caused a 28% increase in iron deposits per cell. Consequently, the thermal doses (cumulative equivalent minutes at 43 °C, CEM43) increased by 15-23% as compared to heat dose achieved for cells treated with magnetic hyperthermia without using enzymes. Likewise, heat-induced cell death increased. In in vivo studies, hyaluronidase-enhanced infiltration and distribution of the nanoparticles in the tumors resulted in moderate heating levels (CEM43 of 128 min as compared to 479 min) and a slower, but persistent decrease in tumor volumes over time after treatment, as compared to comparable treatment without hyaluronidase. The results indicate that hyaluronidase, in particular, improves the infiltration of magnetic nanoparticles into pancreatic cancer models, impacts their thermal treatment and cell depletion, and hence, will contribute immensely in the fight against pancreatic and many other adenocarcinomas.

MNP Enhanced Microwave Imaging by Means of Pseudo-Noise Sensing with Different External Magnetic Field Modulations.

This contribution deals with the detection and imaging of magnetic modulated nanoparticles by means of ultra-wideband sensing. We performed phantom measurements in a practical measurement setup where the magnetic nanoparticles are modulated by a static and a low periodic changing external magnetic field. We investigated the influence of the modulation type of the polarizing magnetic field on both, detectability and imaging of magnetic nanoparticles. We can conclude that both modulations generate a sufficient contrast in order that the nanoparticles were detected at the correct position in a three-dimensional volume. The imaging results, including 32 channels, indicate that the two state (ON/OFF) modulation of the magnetic field under constant environmental conditions shows better results compared to a sinusoidal excitation of the magnetic field.

Rapid Target Binding and Cargo Release of Activatable Liposomes Bearing HER2 and FAP Single-Chain Antibody Fragments Reveal Potentials for Image-Guided Delivery to Tumors.

Liposomes represent suitable tools for the diagnosis and treatment of a variety of diseases, including cancers. To study the role of the human epidermal growth factor receptor 2 (HER2) as target in cancer imaging and image-guided deliveries, liposomes were encapsulated with an intrinsically quenched concentration of a near-infrared fluorescent dye in their aqueous interior. This resulted in quenched liposomes (termed LipQ), that were fluorescent exclusively upon degradation, dye release, and activation. The liposomes carried an always-on green fluorescent phospholipid in the lipid layer to enable tracking of intact liposomes. Additionally, they were functionalized with single-chain antibody fragments directed to fibroblast activation protein (FAP), a marker of stromal fibroblasts of most epithelial cancers, and to HER2, whose overexpression in 20-30% of all breast cancers and many other cancer types is associated with a poor treatment outcome and relapse. We show that both monospecific (HER2-IL) and bispecific (Bi-FAP/HER2-IL) formulations are quenched and undergo HER2-dependent rapid uptake and cargo release in cultured target cells and tumor models in mice. Thereby, tumor fluorescence was retained in whole-body NIRF imaging for 32-48 h post-injection. Opposed to cell culture studies, Bi-FAP/HER2-IL-based live confocal microscopy of a high HER2-expressing tumor revealed nuclear delivery of the encapsulated dye. Thus, the liposomes have potentials for image-guided nuclear delivery of therapeutics, and also for intraoperative delineation of tumors, metastasis, and tumor margins.

The Treatment of Heterotopic Human Colon Xenograft Tumors in Mice with 5-Fluorouracil Attached to Magnetic Nanoparticles in Combination with Magnetic Hyperthermia Is More Efficient than Either Therapy Alone.

Magnetic nanoparticles (MNPs) have shown promising features to be utilized in combinatorial magnetic hyperthermia and chemotherapy. Here, we assessed if a thermo-chemotherapeutic approach consisting of the intratumoral application of functionalized chitosan-coated MNPs (CS-MNPs) with 5-fluorouracil (5FU) and magnetic hyperthermia prospectively improves the treatment of colorectal cancer. With utilization of a human colorectal cancer (HT29) heterotopic tumor model in mice, we showed that the thermo-chemotherapeutic treatment is more efficient in inactivating colon cancer than either tumor treatments alone (i.e., magnetic hyperthermia vs. the presence of 5FU attached to MNPs). In particular, the thermo-chemotherapeutic treatment significantly (p < 0.01) impacts tumor volume and tumor cell proliferation (Ki67 expression, p < 0.001) compared to the single therapy modalities. The thermo-chemotherapeutic treatment: (a) affects DNA replication and repair as measured by H2AX and phosphorylated H2AX expression (p < 0.05 to 0.001), (b) it does not distinctly induce apoptosis nor necroptosis in target cells, since expression of p53, PARP cleaved-PARP, caspases and phosphorylated-RIP3 was non-conspicuous, (c) it renders tumor cells surviving therapy more sensitive to further therapy sessions as indicated by an increased expression of p53, reduced expression of NF-κB and HSPs, albeit by tendency with p > 0.05), and (d) that it impacts tumor vascularity (reduced expression of CD31 and αvß3 integrin (p < 0.01 to 0.001) and consequently nutrient supply to tumors. We further hypothesize that tumor cells die, at least in parts, via a ROS dependent mechanism called oxeiptosis. Taken together, a very effective elimination of colon cancers seems to be feasible by utilization of repeated thermo-chemotherapeutic therapy sessions in the long-term.

Iron Oxide Nanoparticles as Carriers for DOX and Magnetic Hyperthermia after Intratumoral Application into Breast Cancer in Mice: Impact and Future Perspectives.

There is still a need for improving the treatment of breast cancer with doxorubicin (DOX). In this paper, we functionalized magnetic nanoparticles (MNPs) with DOX and studied the DOX-induced antitumor effects in breast cancer cells (BT474) in the presence of magnetic hyperthermia (43 °C, 1 h). We show that i) intratumoral application of DOX-functionalized MNPs (at least at a concentration of 9.6 nmol DOX/100 mm3 tumor volume) combined with magnetic hyperthermia favors tumor regression in vivo, and there is evidence for an increased effect compared to magnetic hyperthermia alone or to the intratumoral application of free DOX and ii) the presence of the pseudopeptide NucAnt (N6L) on the MNP surface might well be beneficial in its function as carrier for MNP internalization into breast cancer cells in vitro, which could further augment the possibility of the induction of intracellular heating spots and cell death in the future.

Targeting the Tumor Microenvironment with Fluorescence-Activatable Bispecific Endoglin/Fibroblast Activation Protein Targeting Liposomes.

Liposomes are biocompatible nanocarriers with promising features for targeted delivery of contrast agents and drugs into the tumor microenvironment, for imaging and therapy purposes. Liposome-based simultaneous targeting of tumor associated fibroblast and the vasculature is promising, but the heterogeneity of tumors entails a thorough validation of suitable markers for targeted delivery. Thus, we elucidated the potential of bispecific liposomes targeting the fibroblast activation protein (FAP) on tumor stromal fibroblasts, together with endoglin which is overexpressed on tumor neovascular cells and some neoplastic cells. Fluorescence-quenched liposomes were prepared by hydrating a lipid film with a high concentration of the self-quenching near-infrared fluorescent dye, DY-676-COOH, to enable fluorescence detection exclusively upon liposomal degradation and subsequent activation. A non-quenched green fluorescent phospholipid was embedded in the liposomal surface to fluorescence-track intact liposomes. FAP- and murine endoglin-specific single chain antibody fragments were coupled to the liposomal surface, and the liposomal potentials validated in tumor cells and mice models. The bispecific liposomes revealed strong fluorescence quenching, activatability, and selectivity for target cells and delivered the encapsulated dye selectively into tumor vessels and tumor associated fibroblasts in xenografted mice models and enabled their fluorescence imaging. Furthermore, detection of swollen lymph nodes during intra-operative simulations was possible. Thus, the bispecific liposomes have potentials for targeted delivery into the tumor microenvironment and for image-guided surgery.