Intermittent Fasting Primes the Tumor Microenvironment and Improves Nanomedicine Delivery in Hepatocellular Carcinoma.

Fasting has many health benefits, including reduced chemotherapy toxicity and improved efficacy. It is unclear how fasting affects the tumor microenvironment (TME) and tumor-targeted drug delivery. Here the effects of intermittent (IF) and short-term (STF) fasting are investigated on tumor growth, TME composition, and liposome delivery in allogeneic hepatocellular carcinoma (HCC) mouse models. To this end, mice are inoculated either subcutaneously or intrahepatically with Hep-55.1C cells and subjected to IF for 24 d or to STF for 1 d. IF but not STF significantly slows down tumor growth. IF increases tumor vascularization and decreases collagen density, resulting in improved liposome delivery. In vitro, fasting furthermore promotes the tumor cell uptake of liposomes. These results demonstrate that IF shapes the TME in HCC towards enhanced drug delivery. Finally, when combining IF with liposomal doxorubicin treatment, the antitumor efficacy of nanochemotherapy is found to be increased, while systemic side effects are reduced. Altogether, these findings exemplify that the beneficial effects of fasting on anticancer therapy outcomes go beyond modulating metabolism at the molecular level.

Chemokine CXCL10 Modulates the Tumor Microenvironment of Fibrosis-Associated Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) constitutes a devastating health burden. Recently, tumor microenvironment-directed interventions have profoundly changed the landscape of HCC therapy. In the present study, the function of the chemokine CXCL10 during fibrosis-associated hepatocarcinogenesis was analyzed with specific focus on its impact in shaping the tumor microenvironment. C57BL/6J wild type (WT) and Cxcl10 knockout mice (Cxcl10-/-) were treated with diethylnitrosamine (DEN) and tetrachloromethane (CCl4) to induce fibrosis-associated HCCs. Cxcl10 deficiency attenuated hepatocarcinogenesis by decreasing tumor cell proliferation as well as tumor vascularization and modulated tumor-associated extracellular matrix composition. Furthermore, the genetic inactivation of Cxcl10 mediated an alteration of the tumor-associated immune response and modified chemokine/chemokine receptor networks. The DEN/CCl4-treated Cxcl10-/- mice presented with a pro-inflammatory tumor microenvironment and an accumulation of anti-tumoral immune cells in the tissue. The most striking alteration in the Cxcl10-/- tumor immune microenvironment was a vast accumulation of anti-tumoral T cells in the invasive tumor margin. In summary, our results demonstrate that CXCL10 exerts a non-redundant impact on several hallmarks of the tumor microenvironment and especially modulates the infiltration of anti-tumorigenic immune cells in HCC. In the era of microenvironment-targeted HCC therapies, interfering with CXCL10 defines a novel asset for further improvement of therapeutic strategies.

RGD-coated polymeric microbubbles promote ultrasound-mediated drug delivery in an inflamed endothelium-pericyte co-culture model of the blood-brain barrier.

Drug delivery to central nervous pathologies is compromised by the blood-brain barrier (BBB). A clinically explored strategy to promote drug delivery across the BBB is sonopermeation, which relies on the combined use of ultrasound (US) and microbubbles (MB) to induce temporally and spatially controlled opening of the BBB. We developed an advanced in vitro BBB model to study the impact of sonopermeation on the delivery of the prototypic polymeric drug carrier pHPMA as a larger molecule and the small molecule antiviral drug ribavirin. This was done under standard and under inflammatory conditions, employing both untargeted and RGD peptide-coated MB. The BBB model is based on human cerebral capillary endothelial cells and human placental pericytes, which are co-cultivated in transwell inserts and which present with proper transendothelial electrical resistance (TEER). Sonopermeation induced a significant decrease in TEER values and facilitated the trans-BBB delivery of fluorescently labeled pHPMA (Atto488-pHPMA). To study drug delivery under inflamed endothelial conditions, which are typical for e.g. tumors, neurodegenerative diseases and CNS infections, tumor necrosis factor (TNF) was employed to induce inflammation in the BBB model. RGD-coated MB bound to and permeabilized the inflamed endothelium-pericyte co-culture model, and potently improved Atto488-pHPMA and ribavirin delivery. Taken together, our work combines in vitro BBB bioengineering with MB-mediated drug delivery enhancement, thereby providing a framework for future studies on optimization of US-mediated drug delivery to the brain.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

Repeated Contrast-Enhanced Micro-CT Examinations Decrease Animal Welfare and Influence Tumor Physiology.

OBJECTIVES: Computed tomography (CT) imaging is considered relatively safe and is often used in preclinical research to study physiological processes. However, the sum of low-dose radiation, anesthesia, and animal handling might impact animal welfare and physiological parameters. This is particularly relevant for longitudinal studies with repeated CT examinations. Therefore, we investigated the influence of repeated native and contrast-enhanced (CE) CT on animal welfare and tumor physiology in regorafenib-treated and nontreated tumor-bearing mice. MATERIAL AND METHODS: Mice bearing 4T1 breast cancer were divided into 5 groups: (1) no imaging, (2) isoflurane anesthesia only, (3) 4 mGy CT, (4) 50 mGy CT, and (5) CE-CT (iomeprol). In addition, half of each group was treated with the multikinase inhibitor regorafenib. Mice were imaged 3 times within 1 week under isoflurane anesthesia. Behavioral alterations were investigated by score sheet evaluation, rotarod test, heart rate measurements, and fecal corticosterone metabolite analysis. Tumor growth was measured daily with a caliper. Tumors were excised at the end of the experiment and histologically examined for blood vessel density, perfusion, and cell proliferation. RESULTS: According to the score sheet, animals showed a higher burden after anesthesia administration and in addition with CT imaging ( P < 0.001). Motor coordination was not affected by native CT, but significantly decreased after CE-CT in combination with the tumor therapy ( P < 0.001). Whereas tumor growth and blood vessel density were not influenced by anesthesia or imaging, CT-scanned animals had a higher tumor perfusion ( P < 0.001) and a lower tumor cell proliferation ( P < 0.001) for both radiation doses. The most significant difference was observed between the control and CE-CT groups. CONCLUSION: Repeated (CE-) CT imaging of anesthetized animals can lead to an impairment of animal motor coordination and, thus, welfare. Furthermore, these standard CT protocols seem to be capable of inducing alterations in tumor physiology when applied repetitively. These potential effects of native and CE-CT should be carefully considered in preclinical oncological research.

Poly-l-glutamic acid modification modulates the bio-nano interface of a therapeutic anti-IGF-1R antibody in prostate cancer.

Modifying biological agents with polymers such as polyethylene glycol (PEG) has demonstrated clinical benefits; however, post-market surveillance of PEGylated derivatives has revealed PEG-associated toxicity issues, prompting the search for alternatives. We explore how conjugating a poly-l-glutamic acid (PGA) to an anti-insulin growth factor 1 receptor antibody (AVE1642) modulates the bio-nano interface and anti-tumor activity in preclinical prostate cancer models. Native and PGA-modified AVE1642 display similar anti-tumor activity in vitro; however, AVE1642 prompts IGF-1R internalization while PGA conjugation prompts higher affinity IGF-1R binding, thereby inhibiting IGF-1R internalization and altering cell trafficking. AVE1642 attenuates phosphoinositide 3-kinase signaling, while PGA-AVE1642 inhibits phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. PGA conjugation also enhances AVE1642's anti-tumor activity in an orthotopic prostate cancer mouse model, while PGA-AVE1642 induces more significant suppression of cancer cell proliferation/angiogenesis than AVE1642. These findings demonstrate that PGA conjugation modulates an antibody's bio-nano interface, mechanism of action, and therapeutic activity.

Monitoring EPR Effect Dynamics during Nanotaxane Treatment with Theranostic Polymeric Micelles.

Cancer nanomedicines rely on the enhanced permeability and retention (EPR) effect for efficient target site accumulation. The EPR effect, however, is highly heterogeneous among different tumor types and cancer patients and its extent is expected to dynamically change during the course of nanochemotherapy. Here the authors set out to longitudinally study the dynamics of the EPR effect upon single- and double-dose nanotherapy with fluorophore-labeled and paclitaxel-loaded polymeric micelles. Using computed tomography-fluorescence molecular tomography imaging, it is shown that the extent of nanomedicine tumor accumulation is predictive for therapy outcome. It is also shown that the interindividual heterogeneity in EPR-based tumor accumulation significantly increases during treatment, especially for more efficient double-dose nanotaxane therapy. Furthermore, for double-dose micelle therapy, tumor accumulation significantly increased over time, from 7% injected dose per gram (ID g-1 ) upon the first administration to 15% ID g-1 upon the fifth administration, contributing to more efficient inhibition of tumor growth. These findings shed light on the dynamics of the EPR effect during nanomedicine treatment and they exemplify the importance of using imaging in nanomedicine treatment prediction and clinical translation.

Effects of contrast-enhanced ultrasound treatment on neoadjuvant chemotherapy in breast cancer.

Purpose: Preclinical and clinical data indicate that contrast-enhanced ultrasound can enhance tumor perfusion and vessel permeability, thus, improving chemotherapy accumulation and therapeutic outcome. Therefore, we investigated the effects of high mechanical index (MI) contrast-enhanced Doppler ultrasound (CDUS) on tumor perfusion in breast cancer. Methods: In this prospective study, breast cancer patients were randomly assigned to receive either 18 minutes of high MI CDUS during chemotherapy infusion (n = 6) or chemotherapy alone (n = 5). Tumor perfusion was measured before and after at least six chemotherapy cycles using motion-model ultrasound localization microscopy. Additionally, acute effects of CDUS on vessel perfusion and chemotherapy distribution were evaluated in mice bearing triple-negative breast cancer (TNBC). Results: Morphological and functional vascular characteristics of breast cancer in patients were not significantly influenced by high MI CDUS. However, complete clinical tumor response after neoadjuvant chemotherapy was lower in high MI CDUS-treated (1/6) compared to untreated patients (4/5) and size reduction of high MI CDUS treated tumors tended to be delayed at early chemotherapy cycles. In mice with TNBC high MI CDUS decreased the perfused tumor vessel fraction (p < 0.01) without affecting carboplatin accumulation or distribution. Higher vascular immaturity and lower stromal stabilization may explain the stronger vascular response in murine than human tumors. Conclusion: High MI CDUS had no detectable effect on breast cancer vascularization in patients. In mice, the same high MI CDUS setting did not affect chemotherapy accumulation although strong effects on the tumor vasculature were detected histologically. Thus, sonopermeabilization in human breast cancers might not be effective using high MI CDUS protocols and future applications may rather focus on low MI approaches triggering microbubble oscillations instead of destruction. Furthermore, our results show that there are profound differences in the response of mouse and human tumor vasculature to high MI CDUS, which need to be further explored and considered in clinical translation.

Influence of MRI Examinations on Animal Welfare and Study Results.

OBJECTIVES: Magnetic resonance imaging (MRI) is considered to be well tolerated by laboratory animals. However, no systematic study has been performed yet, proving this assumption. Therefore, the aim of this study was to investigate the possible effects of longitudinal native and contrast-enhanced (CE) 1-T and 7-T MRI examinations on mouse welfare as well as 4T1 breast cancers progression and therapy response. MATERIAL AND METHODS: Forty-seven healthy and 72 breast cancer-bearing mice (4T1) were investigated. One-Tesla (ICON) and 7-T (Biospec) MRI measurements were performed thrice per week under isoflurane anesthesia in healthy BALB/c mice for 4 weeks and 3 times within 2 weeks in tumor-bearing animals. Animal welfare was examined by an observational score sheet, rotarod performance, heart rate measurements, and assessment of fecal corticosterone metabolites. Furthermore, we investigated whether CE-MRI influences the study outcome. Therefore, hemograms and organ weights were obtained, and 4T1 tumor growth, perfusion, immune cell infiltration, as well as response to the multikinase inhibitor regorafenib were investigated. Statistical comparisons between groups were performed using analysis of variance and Tukey or Bonferroni post hoc tests. RESULTS: Mice showed no alterations in the observational score sheet rating, rotarod performance, heart rate, and fecal corticosterone metabolites (P > 0.05) after repeated MRI at both field strengths. However, spleen weights were reduced in all healthy mouse groups that received isoflurane anesthesia (P < 0.001) including the groups investigated by 1-T and 7-T MRI (P = 0.02). Neither tumor progression nor response to the regorafenib treatment was affected by isoflurane anesthesia or CE-MRI monitoring. Furthermore, immunohistological tumor analysis did not indicate an effect of isoflurane and MRI on macrophage infiltration of tumors, perfusion of tumor vessels, and apoptotic cell rate (P > 0.05). CONCLUSIONS: Repeated MRI did not influence the welfare of mice and did not affect tumor growth and therapy response of 4T1 tumors. However, systemic immunological effects of isoflurane anesthesia need to be considered to prevent potential bias.

Multimodal and multiscale optical imaging of nanomedicine delivery across the blood-brain barrier upon sonopermeation.

Rationale: The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. Sonopermeation, which relies on the combination of ultrasound and microbubbles, has emerged as a powerful tool to permeate the BBB, enabling the extravasation of drugs and drug delivery systems (DDS) to and into the central nervous system (CNS). When aiming to improve the treatment of high medical need brain disorders, it is important to systematically study nanomedicine translocation across the sonopermeated BBB. To this end, we here employed multimodal and multiscale optical imaging to investigate the impact of DDS size on brain accumulation, extravasation and penetration upon sonopermeation. Methods: Two prototypic DDS, i.e. 10 nm-sized pHPMA polymers and 100 nm-sized PEGylated liposomes, were labeled with fluorophores and intravenously injected in healthy CD-1 nude mice. Upon sonopermeation, computed tomography-fluorescence molecular tomography, fluorescence reflectance imaging, fluorescence microscopy, confocal microscopy and stimulated emission depletion nanoscopy were used to study the effect of DDS size on their translocation across the BBB. Results: Sonopermeation treatment enabled safe and efficient opening of the BBB, which was confirmed by staining extravasated endogenous IgG. No micro-hemorrhages, edema and necrosis were detected in H&E stainings. Multimodal and multiscale optical imaging showed that sonopermeation promoted the accumulation of nanocarriers in mouse brains, and that 10 nm-sized polymeric DDS accumulated more strongly and penetrated deeper into the brain than 100 nm-sized liposomes. Conclusions: BBB opening via sonopermeation enables safe and efficient delivery of nanomedicine formulations to and into the brain. When looking at accumulation and penetration (and when neglecting issues such as drug loading capacity and therapeutic efficacy) smaller-sized DDS are found to be more suitable for drug delivery across the BBB than larger-sized DDS. These findings are valuable for better understanding and further developing nanomedicine-based strategies for the treatment of CNS disorders.

Tumor targeting via EPR: Strategies to enhance patient responses.

The tumor accumulation of nanomedicines relies on the enhanced permeability and retention (EPR) effect. In the last 5-10 years, it has been increasingly recognized that there is a large inter- and intra-individual heterogeneity in EPR-mediated tumor targeting, explaining the heterogeneous outcomes of clinical trials in which nanomedicine formulations have been evaluated. To address this heterogeneity, as in other areas of oncology drug development, we have to move away from a one-size-fits-all tumor targeting approach, towards methods that can be employed to individualize and improve nanomedicine treatments. To this end, efforts have to be invested in better understanding the nature, the complexity and the heterogeneity of the EPR effect, and in establishing systems and strategies to enhance, combine, bypass and image EPR-based tumor targeting. In the present manuscript, we summarize key studies in which these strategies are explored, and we discuss how these approaches can be employed to enhance patient responses.