Intratumoral Distribution Research Articles

Optimization of amino acid-based poly(ester urea urethane) nanoparticles for the systemic delivery of gambogic acid for treating triple negative breast cancer.

Amino acid-based poly(ester urea urethane) (AA-PEUU) is developed from amino acid-based ester urea building blocks interconnected with urethane blocks functionalized with poly(ethylene glycol) (PEG). Each functional block consists of structural design features that could impact the properties and performances of AA-PEUU as a nanocarrier for the systemic delivery of gambogic acid (GA). The multifunctional AA-PEUU structure provides broad tunability to enable the optimization of nanocarriers. The study investigates the structure-property relationship by fine-tuning the structure of AA-PEUU, including the amino acid type, hydrocarbons, the ratio of functional building blocks, and PEGylation, to identify the nanoparticle candidate with optimized delivery performances. Compared to free GA, the optimized PEUU nanocarrier improves the intratumoral distribution of GA by more than 9-fold, which significantly enhances the bioavailability and persistence of GA after intravenous administration. In an MDA-MB-231 xenograft mouse model, GA delivered by the optimized AA-PEUU nanocarrier exhibits significant tumor inhibition, apoptosis induction, and the anti-angiogenesis effect. The study demonstrates the potency of engineering AA-PEUU nanocarriers with tailor-designed structures and versatile tunability for the systemic delivery of therapeutics in the treatment of triple negative breast tumor.

Radiation Therapy Modulates Tumor Physical Characteristics to Reduce Intratumoral Pressure and Enhance Intratumoral Drug Delivery and Retention

PurposeHigh intratumoral pressure, caused by tumor cell-to-cell interactions, interstitial fluid pressure, and surrounding stromal composition, plays a substantial role in resistance to intratumoral drug delivery and distribution. Radiation therapy (XRT) is commonly administered in conjunction with different intratumoral drugs, but assessing how radiation can reduce pressure locally and help intratumoral drug administration and retention is important. Methods and Materials344SQ-parental or 344SQ-anti-programmed cell death protein 1-resistant lung adenocarcinoma cells were established in 129Sv/Ev mice, and irradiated with either 1 Gy × 2, 5 Gy × 3, 8 Gy × 3, 12 Gy × 3, or 20 Gy × 1. Intratumoral pressure was measured every 3 to 4 days after XRT. Contrast dye was injected into the tumors 3- and 6-days after XRT, and imaged to measure drug retention. ResultsIn the 344SQ-parental model, low-dose radiation (1 Gy × 2) created an early window of reduced intratumoral pressure 1 to 3 days after XRT compared with untreated control. High-dose stereotactic radiation (12 Gy × 3) reduced intratumoral pressure 3 to 12 days after XRT, and 20 Gy × 1 showed a delayed pressure reduction on day 12. Intermediate doses of radiation did not significantly affect intratumoral pressure. In the more aggressive 344SQ-anti-programmed cell death protein 1-resistant model, low-dose radiation reduced pressure 1 to 5 days after XRT, and 12 Gy × 3 reduced pressure 1 to 3 days after XRT. Moreover, both 1 Gy × 2 and 12 Gy × 3 significantly improved drug retention 3 days after XRT; however, there was no significance detected 6 days after XRT. Lastly, a histopathologic evaluation showed that 1 Gy × 2 reduced collagen deposition within the tumor, and 12 Gy × 3 led to more necrotic core and higher extracellular matrix formation in the tumor periphery. ConclusionsOptimized low-dose XRT, as well as higher stereotactic XRT regimen led to a reduction in intratumoral pressure and increased drug retention. The findings from this work can be readily translated into the clinic to enhance intratumoral injections of various anticancer agents.

Delivery of aPD-L1 antibody to i.p. tumors via direct penetration by i.p. route: Beyond EPR effect.

Chemotherapy for peritoneal dissemination is poorly effective owing to limited drug transfer from the blood to the intraperitoneal (i.p.) compartment after intravenous (i.v.) administration. i.p. chemotherapy has been investigated to improve drug delivery to tumors; however, the efficacy continues to be debated. As anticancer drugs have low molecular weight and are rapidly excreted through the peritoneal blood vessels, maintaining the i.p. concentration as high as expected is a challenge. In this study, we examined whether i.p. administration is an efficient route of administration of high-molecular-weight immune checkpoint inhibitors (ICIs) for the treatment of peritoneal dissemination using a model of peritoneal disseminated carcinoma. After i.p. administration, the amount of anti-PD-L1 antibody transferred into i.p. tumors increased by approximately eight folds compared to that after i.v. administration. Intratumoral distribution analysis revealed that anti-PD-L1 antibodies were delivered directly from the i.p. space to the surface of tumor tissue, and that they deeply penetrated the tumor tissues after i.p. administration; in contrast, after i.v. administration, anti-PD-L1 antibodies were only distributed around blood vessels in tumor tissues via the enhanced permeability and retention (EPR) effect. Owing to the enhanced delivery, the therapeutic efficacy of anti-PD-L1 antibody in the peritoneal dissemination models was also improved after i.p. administration compared to that after i.v. administration. This is the first study to clearly demonstrate an EPR-independent delivery of ICIs to i.p. tumors by which ICIs were delivered in a massive amount to the tumor tissue via direct penetration after i.p. administration.

Graphene Oxide Nanoscale Platform Enhances the Anti-Cancer Properties of Bortezomib in Glioblastoma Models.

Graphene-based 2D nanomaterials possess unique physicochemical characteristics which can be utilized in various biomedical applications, including the transport and presentation of chemotherapeutic agents. In glioblastoma multiforme (GBM), intratumorally administered thin graphene oxide (GO) nanosheets demonstrate a widespread distribution throughout the tumor volume without impact on tumor growth, nor spread into normal brain tissue. Such intratumoral localization and distribution can offer multiple opportunities for treatment and modulation of the GBM microenvironment. Here, the kinetics of GO nanosheet distribution in orthotopic GBM mouse models is described and a novel nano-chemotherapeutic approach utilizing thin GO sheets as platforms to non-covalently complex a proteasome inhibitor, bortezomib (BTZ), is rationally designed. Through the characterization of the GO:BTZ complexes, a high loading capacity of the small molecule on the GO surface with sustained BTZ biological activity in vitro is demonstrated. In vivo, a single low-volume intratumoral administration of GO:BTZ complex shows an enhanced cytotoxic effect compared to free drug in two orthotopic GBM mouse models. This study provides evidence of the potential that thin and small GO sheets hold as flat nanoscale platforms for GBM treatment by increasing the bioavailable drug concentration locally, leading to an enhanced therapeutic effect.

Multiplexed Imaging Reveals the Spatial Relationship of the Extracellular Acidity-Targeting pHLIP with Necrosis, Hypoxia, and the Integrin-Targeting cRGD Peptide.

pH (low) insertion peptides (pHLIPs) have been developed for cancer imaging and therapy targeting the acidic extracellular microenvironment. However, the characteristics of intratumoral distribution (ITD) of pHLIPs are not yet fully understood. This study aimed to reveal the details of the ITD of pHLIPs and their spatial relationship with other tumor features of concern. The fluorescent dye-labeled pHLIPs were intravenously administered to subcutaneous xenograft mouse models of U87MG and IGR-OV1 expressing αVβ3 integrins (using large necrotic tumors). The αVβ3 integrin-targeting Cy5.5-RAFT-c(-RGDfK-)4 was used as a reference. In vivo and ex vivo fluorescence imaging, whole-tumor section imaging, fluorescence microscopy, and multiplexed fluorescence colocalization analysis were performed. The ITD of fluorescent dye-labeled pHLIPs was heterogeneous, having a high degree of colocalization with necrosis. A direct one-to-one comparison of highly magnified images revealed the cellular localization of pHLIP in pyknotic, karyorrhexis, and karyolytic necrotic cells. pHLIP and hypoxia were spatially contiguous but not overlapping cellularly. The hypoxic region was found between the ITDs of pHLIP and the cRGD peptide and the Ki-67 proliferative activity remained detectable in the pHLIP-accumulated regions. The results provide a better understanding of the characteristics of ITD of pHLIPs, leading to new insights into the theranostic applications of pHLIPs.

Chronic convection-enhanced intratumoural delivery of chemotherapy for glioblastoma.

In The Lancet Oncology, Eleonora F Spinazzi and colleagues1 present a first-in-human study of chronic convection-enhanced delivery (CED) of chemotherapy to five patients with recurrent glioblastoma.1 CED involves stereotactic placement of catheter(s) into a tumour, followed by connecting the catheter(s) to pumps to allow intratumoural drug distribution by bulk flow via continuous, low-grade, positive-pressure microinfusion.2 The rationale for CED of chemotherapies in glioblastoma is the ability of this technique to deliver high intratumoural drug concentrations throughout the tumour, bypass the blood–brain barrier, and avoid toxic effects from systemic drug administration.

Injectable rapidly dissolving needle-type gelatin implant capable of delivering high concentrations of H2O2 through intratumoral injection

Intratumoral injections can reduce systemic absorption and deliver large amounts of drugs to the tumor, thereby reducing side effects and exhibiting high therapeutic efficacy. Therefore, a variety of drug delivery systems, such as hydrogels, fine particles, and nanoparticles, have been studied. Although the sustained-release drug delivery system can effectively reduce systemic absorption due to the slow release of the drug from the site of intratumoral injection, it lacks the ability to deliver high concentrations of drugs to the tumor. In particular, the larger the tumor size, the lower the efficacy of the treatment. To address this problem, this study focused on the tumor structure. Owing to the three-dimensional structure of the dense tumor microenvironment (TME) and abnormal blood vessels, drugs administered directly into the tumor act as if they were encapsulated in a hydrogel. To evaluate whether the three-dimensional structure of the tumor affects the intratumoral distribution and systemic absorption of drugs, needle-type starch implants (GOD-NS implants) and needle-type gelatin implants (GOD-NG implants) containing glucose oxidase (GOD), a protein that exhibits anti-cancer effects through hydrogen peroxide (H2O2) generation, were prepared. Both GOD-NS and GOD-NG implants can be easily injected into tumors. GOD-NS implants released GOD slowly, whereas GOD-NG implants released most of the GOD within 1 h. When a GOD-NG implant that rapidly released GOD was also injected, a high concentration of GOD was maintained in the tumor for a long time as it was trapped in the three-dimensional structure of the tumor. This study demonstrated that intratumoral injection of a rapidly drug-releasing gelatin needle may be a novel drug delivery system capable of long-term retention of high drug concentrations in tumors, as the three-dimensional structure of the tumor affects drug delivery.

Microvesicle-inspired oxygen-delivering nanosystem potentiates radiotherapy-mediated modulation of tumor stroma and antitumor immunity

The efficacy of radiotherapy is greatly challenged by intense hypoxia, intricate stroma and suppressive immune microenvironments in tumors. Herein, we rationally designed a microvesicle-inspired oxygen-delivering polyfluorocarbon nanosystem loading DiIC18(5) and halofuginone (M-FDH) with prominent capacity of improving tumor oxygenation and intratumor distribution, synergizing radiation to disrupt tumor stroma and boost antitumor immunity for combinational cancer therapy. M-FDH produced a 10.98-fold enhancement of tumor oxygenation and caused efficient production of reactive oxygen species (ROS) upon radiation. M-FDH + X ray treatment resulted in notable DNA damages, over 90% elimination of cancer-associated fibroblasts (CAFs) and major components of extracellular matrix, significant enhancement of tumoricidal CD3+CD8+ T cells, and profound elimination of suppressive immune cells in 4T1 tumors. The therapeutic benefits of M-FDH + X ray on suppressing tumor growth were confirmed in two murine tumor models. Therefore, this study provides an encouraging microvesicle-inspired strategy to target cancer cells and CAFs in tumors and synergize radiotherapy for effective cancer treatment.

Pharmacokinetics and Pharmacodynamics of TAK-164 Antibody Drug Conjugate Coadministered with Unconjugated Antibody.

The development of new antibody-drug conjugates (ADCs) has led to the approval of 7 ADCs by the FDA in 4years. Given the impact of intratumoral distribution on efficacy of these therapeutics, coadministration of unconjugated antibody with ADC has been shown to improve distribution and efficacy of several ADCs in high and moderately expressed tumor target systems by increasing tissue penetration. However, the benefit of coadministration in low expression systems is less clear. TAK-164, an ADC composed of an anti-GCC antibody (5F9) conjugated to a DGN549 payload, has demonstrated heterogeneous distribution and bystander killing. Here, we evaluated the impact of 5F9 coadministration on distribution and efficacy of TAK-164 in a primary human tumor xenograft mouse model. Coadministration was found to improve the distribution of TAK-164 within the tumor, but it had no significant impact (increase or decrease) on efficacy. Experimental and computational evidence indicates that this was not a result of tumor saturation, increased binding to perivascular cells, or compensatory bystander effects. Rather, the cellular potency of DGN549 was matched with the single-cell uptake of TAK-164 making its IC50 close to its equilibrium binding affinity (KD), and as such, coadministration dilutes total DGN549 in cells below the maximum cytotoxic concentration, thereby offsetting an increased number of targeted cells with decreased ability to kill each cell. These results provide new insights on matching payload potency to ADC delivery to help identify when increasing tumor penetration is beneficial for improving ADC efficacy and demonstrate how mechanistic simulations can be leveraged to design clinically effective ADCs.

Spatial Transcriptomics-Based Identification of Molecular Markers for Nanomedicine Distribution in Tumor Tissue.

The intratumoral accumulation of nanomedicine has been considered a passive process, referred to as the enhanced permeability and retention effect. Recent studies have suggested that the tumor uptake of nanomedicines follows an energy-dependent pathway rather than being a passive process. Herein, to explore the factor candidates that are associated with nanomedicine tumor uptake, a molecular marker identification platform is developed by integrating microscopic fluorescence images of a nanomedicine distribution with spatial transcriptomics information. When this approach is applied to PEGylated liposomes, molecular markers related to hypoxia, glycolysis, and apoptosis can be identified as being related to the intratumoral distribution of the nanomedicine. It is expected that the method can be applied to explain the distribution of a wide range of nanomedicines and that the data obtained from this analysis can enhance the precise utilization of nanomedicines.

Impact of tumoral structure and bacterial species on growth and biodistribution of live bacterial therapeutics in xenografted tumours

Live bacterial therapeutics is gaining attention, especially for cancer therapy, because anaerobic bacteria selectively grow inside the solid tumours. However, the effect of tumour structure and bacterial characteristics on the pharmacokinetics of tumours is unclear; therefore, we aimed to elucidate the effects of tumour structure and types of bacteria on tumoral bacterial growth. Using six mouse xenograft models, including stroma-rich tumours similar to clinical tumours, and two models of live bacterial therapeutics, Salmonella typhimurium VNP20009 and Escherichia coli DH5α, we investigated bacterial growth and distribution in tumours after intravenous administration. Rapid growth of E. coli was observed in HCT116 and other tumours with few collagens, blood vessels not covered by mural cells, and a cancer cell area proliferated disorderly, whereas tumours with contrasting features, such as BxPC-3, showed lower bacterial growth and a limited intratumor distribution. Alternatively, Salmonella typhimurium VNP20009, when successfully proliferated (the probability was approximately 50%), grew to 108 colony forming units/g tissue even in BxPC-3 tumours, and its intratumor distribution was extensive. This study suggests that the development of new methods to modify tumour structure will be essential for the development of anti-tumour clinical therapies based on live bacterial therapeutics.

Development of Injectable and Biodegradable Needle-Type Starch Implant for Effective Intratumoral Drug Delivery and Distribution.

IntroductionCompared to intravenous administration, intratumoral drug administration enables the direct delivery of drugs to tumors and mitigates the systemic absorption of drugs and associated drug-induced side effects. However, intratumoral drug administration presents several challenges. The high interstitial fluid pressure (IFP) of the tumor prevents the retention of drugs within the tumor; thus, significant amounts of the drugs are absorbed systemically through the bloodstream or delivered to non-target sites. To solve this problem, in this study, a drug-enclosed needle-type starch implant was developed that can overcome IFP and remain in the tumor.MethodsInjectable needle-type starch implants (NS implants) were prepared by starch gelatinization and drying. The structure, cytotoxicity, and anticancer effects of the NS implants were evaluated. Biodistribution of NS implants was evaluated in pork (in vitro), dissected liver (ex vivo), and 4T1 tumors in mice (in vivo) using a fluorescence imaging device.ResultsThe prepared NS implants exhibited a hydrogel structure after water absorption. NS implants showed effective cytotoxicity and anticancer effects by photothermal therapy (PTT). The NS implant itself has sufficient strength and can be easily injected into a desired area. In vivo, the NS implant continuously delivered drugs to the tumor more effectively and uniformly than conventional hydrogels and solutions.ConclusionThis study demonstrated the advantages of needle-type implants. An injectable NS implant can be a new formulation that can effectively deliver drugs and exhibit anticancer effects.

Multifunctional nanoprobe for real-time in vivo monitoring of T cell activation

Genetically engineered T cells are a powerful new modality for cancer immunotherapy. However, their clinical application for solid tumors is challenging, and crucial knowledge on cell functionality in vivo is lacking. Here, we fabricated a nanoprobe composed of dendrimers incorporating a calcium sensor and gold nanoparticles, for dual-modal monitoring of engineered T cells within a solid tumor. T cells engineered to express a melanoma-specific T-cell receptor and loaded with the nanoprobe were longitudinally monitored within melanoma xenografts in mice. Fluorescent imaging of the nanoprobe's calcium sensor revealed increased intra-tumoral activation of the T cells over time, up to 24 h. Computed tomography imaging of the nanoprobe's gold nanoparticles revealed the cells' intra-tumoral distribution pattern. Quantitative analysis revealed the intra-tumoral T cell quantities. Thus, this nanoprobe reveals intra-tumoral persistence, penetration and functional status of genetically engineered T cells, which can advance T cell-based immunotherapy and promote next-generation live cell imaging.

Frequently expressed glypican-3 as a promising novel therapeutic target for osteosarcomas.

Osteosarcoma (OS) is the most common bone malignancy without a reliable therapeutic target. Glypican‐3 (GPC3) mutation and upregulation have been detected in multidrug resistant OS, and anti‐GPC3 immunotherapy can effectively suppress the growth of organoids. Further profiling of GPC3 mutations and expression patterns in OS is of clinical significance. To address these issues, fresh OS specimens were collected from 24 patients for cancer‐targeted next‐generation sequencing (NGS) and three‐dimensional patient‐derived organoid (PDO) culture. A tumor microarray was prepared using 37 archived OS specimens. Immunohistochemical (IHC) staining was performed on OS specimens and microarrays to profile GPC3 and CD133 expression as well as intratumoral distribution patterns. RT‐PCR was conducted to semiquantify GPC3 and CD133 expression levels in the OS tissues. Anti‐GPC3 immunotherapy was performed on OS organoids with or without GPC3 expression and its efficacy was analyzed using multiple experimental approaches. No OS cases with GPC3 mutations were found, except for the positive control (OS‐08). IHC staining revealed GPC3 expression in 73.77% (45/61) of OSs in weak (+; 29/45), moderate (++; 8/45), and strong (+++; 8/45) immunolabeling densities. The intratumoral distribution of GPC3‐positive cells was variable in the focal (+; 10%–30%; 8/45), partial (++; 31%–70%; 22/45), and the most positive patterns (+++; >71%; 15/45), which coincided with CD133 immunolabeling (P = 9.89 × 10−10). The anti‐GPC3 antibody efficiently inhibits Wnt/β‐catenin signaling and induces apoptosis in GPC3‐positive PDOs and PDXs, as opposed to GPC3‐negative PDOs and PDXs. The high frequency of GPC3 and CD133 co‐expression and the effectiveness of anti‐wild‐type GPC3‐Ab therapy in GPC3‐positive OS models suggest that GPC3 is a novel prognostic parameter and a promising therapeutic target for osteosarcoma.

Enzyme-responsive nano-drug delivery system for combined antitumor therapy

Efficient drug loading, tumor targeting, intratumoral penetration, and cellular uptake are the main factors affecting the effectiveness of drug delivery systems in oncotherapy. Based on the tumor microenvironment, we proposed to develop Curcumin (Cur)-loaded matrix metalloproteinase (MMP)-responsive nanoparticles (Cur-P-NPs) by static electricity, to enhance tumor targeting, cellular uptake, and drug loading efficiency. These nanoparticles combine the properties of both PEG-peptides (cleaved peptide + penetrating peptide) and star-shaped polyester (DPE-PCL) nanoparticles. Cur-P-NPs displayed good entrapment efficiency, drug loading and biocompatibility. Additionally, they showed an enhanced release rate, cellular uptake, and anti-proliferative activity by activating peptides under the simulated tumor microenvironment. Furthermore, intraperitoneal injection of losartan (LST) successfully enhanced intratumoral drug penetration by collagen I degradation. In vivo studies based on the systematic administration of the synergistic LST + Cur-P-NPs combination to mice confirmed that combined antitumor therapy with LST and Cur-P-NPs could further improve intratumor distribution, enhance anticancer efficacy, and reduce the toxicity and side effects. Therefore, LST + Cur-P-NPs represent a new and efficient system for clinical oncotherapy.

Transient Inhibition of Trastuzumab-Tumor Binding to Overcome the "Binding-Site Barrier" and Improve the Efficacy of a Trastuzumab-Gelonin Immunotoxin.

We have recently shown that coadministration of mAbs with anti-idiotypic distribution enhancers (AIDE) that inhibit mAb binding to tumor antigens enabled increased intratumoral mAb distribution and increased efficacy of an antibody-drug conjugate (trastuzumab emtansine, T-DM1). In this article, a pharmacokinetic/pharmacodynamic (PK/PD) model was applied to predict the impact of this optimization strategy on the within-tumor distribution and antitumor efficacy of trastuzumab-gelonin, where the released payload (gelonin) is expected to exhibit negligible bystander activity. Immunofluorescence histology was used to investigate trastuzumab-gelonin distribution in solid tumors following dosing with or without coadministration of anti-trastuzumab AIDEs. Antitumor efficacy of trastuzumab-gelonin, with or without coadministration of AIDEs, was also evaluated in tumor-bearing mice. Trastuzumab-gelonin efficiently induced cytotoxicity when applied to NCI-N87 cells in culture (IC50: 0.224 ± 0.079 nmol/L). PK/PD simulations predicted that anti-idiotypic single-domain antibodies AIDEs with dissociation rate constants between 0.03 and 0.2 per hour would provide optimal enhancement of trastuzumab-gelonin efficacy. LE8 and 1HE, anti-trastuzumab AIDEs, were selected for evaluation in vivo. Coadministration of trastuzumab-gelonin with the inhibitors increased the portion of tumor area that stained positive for trastuzumab-gelonin by 58% (P = 0.0059). In addition, LE8 or 1HE coadministration improved trastuzumab-gelonin efficacy in NCI-N87 xenograft-bearing mice by increasing the percent increase in life span (%ILS) from 27.8% (for trastuzumab-gelonin administered alone) to 62.5% when administered with LE8 (P = 0.0007) or 83.3% (P = 0.0007) when administered with 1HE. These findings support the hypothesis that transient, competitive inhibition of mAb-tumor binding can improve the intratumoral distribution and efficacy of immunotoxins when applied for treatment of solid tumors.

Vascular Remodeling Is a Crucial Event in the Early Phase of Hepatocarcinogenesis in Rodent Models for Liver Tumorigenesis.

Simple SummaryHepatocellular carcinoma (HCC) is a highly vascularized tumor and remodeling of the tumor vasculature is one of the hallmarks of tumor progression. Mouse models are elegant tools to study the onset and progression of liver tumors. However, only few data exist on the vasculature and vascular remodeling processes especially in the early phase of hepatocarcinogenesis. The aim of this study was therefore to perform a comprehensive characterization and comparison of the vasculature in mouse models used for hepatocarcinogenesis studies. For this purpose, we characterized the preneoplastic foci of cellular alteration (FCA) and hepatocellular carcinoma (HCC) by using tissue-based techniques and computer-assisted analysis to better understand if and how vascular remodeling appears in rodent models for liver tumorigenesis. Our findings demonstrated crucial differences in the number and size of the vessels, degree of maturation and intratumoral localization of the vasculature in FCA and HCC, clearly indicating that vascular remodeling is an important step in the early phase of liver tumorigenesis of rodent models.The investigation of hepatocarcinogenesis is a major field of interest in oncology research and rodent models are commonly used to unravel the pathophysiology of onset and progression of hepatocellular carcinoma. HCC is a highly vascularized tumor and vascular remodeling is one of the hallmarks of tumor progression. To date, only a few detailed data exist about the vasculature and vascular remodeling in rodent models used for hepatocarcinogenesis. In this study, the vasculature of HCC and the preneoplastic foci of alteration (FCA) of different mouse models with varying genetic backgrounds were comprehensively characterized by using immunohistochemistry (CD31, Collagen IV, αSMA, Desmin and LYVE1) and RNA in situ hybridization (VEGF-A). Computational image analysis was performed to evaluate selected parameters including microvessel density, pericyte coverage, vessel size, intratumoral vessel distribution and architecture using the Aperio ImageScope and Definiens software programs. HCC presented with a significantly lower number of vessels, but larger vessel size and increased coverage, leading to a higher degree of maturation, whereas FCA lesions presented with a higher microvessel density and a higher amount of smaller but more immature vessels. Our results clearly demonstrate that vascular remodeling is present and crucial in early stages of experimental hepatocarcinogenesis. In addition, our detailed characterization provides a strong basis for further angiogenesis studies in these experimental models.

Tumour immune microenvironment in resected thymic carcinomas as a predictor of clinical outcome.

The spatial distribution of tumour-infiltrating lymphocytes (TILs) is a novel descriptor characterising the tumour immune microenvironment (TIME). The aim of our study was to assess whether a specific TIME of surgically resected thymic carcinoma (TC) can predict tumour invasiveness, recurrence or survival. Digital microscopy was performed on 39 TCs immunohistochemically stained to investigate the activation of the immune checkpoint pathway (PD-L1/PD-1), along with density and spatial distribution of TILs phenotypes (CD3+, CD4+, CD8+, FOXP3+, CD56+). The impact of PD-L1 and TIL density considering the intratumoural (iTILs) and stromal (sTILs) distribution on pathological characteristics and clinical outcomes were analysed. In early TC stages, we observed a higher total density of CD3+ (p = 0.05) and CD8+ (p = 0.02) TILs. PD-L1 was expressed in 71.8% of TCs. In advanced TC stages, we observed a lower density of CD3+ (p = 0.04) and CD8+ (p = 0.01) iTILs compared to early stages. Serum concentrations of PD-L1 were significantly higher in TCs compared to healthy controls: 134.43 ± 18.51 vs. 82.01 ± 6.34 pg/ml (p = 0.001), respectively. High densities of stromal CD4+ TILs (54 vs. 32%, p = 0.043) and CD8+ TILs (65 vs. 17%, p = 0.048) were associated with improved freedom from recurrence (FFR) and cause-specific survival (CSS). High density of FoxP3+ TILs were associated with improved FFR (p = 0.03) and CSS (p = 0.003). Mapping TIL subpopulations complement the armamentarium for prognostication of TC outcomes. The improved outcome in patients with high density of TILs supports the use of immune checkpoint inhibitors in TC patients.

Spatiotemporal analysis of glioma heterogeneity reveals COL1A1 as an actionable target to disrupt tumor progression

Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. However, the mechanisms that set up tumor heterogeneity and tumor cell migration remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a specific, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and potentially the collective invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams structure and function depend on overexpression of COL1A1. Col1a1 is a central gene in the dynamic organization of glioma mesenchymal transformation, and a powerful regulator of glioma malignant behavior. Inhibition of Col1a1 eliminates oncostreams, reprograms the malignant histopathological phenotype, reduces expression of the mesenchymal associated genes, induces changes in the tumor microenvironment and prolongs animal survival. Oncostreams represent a pathological marker of potential value for diagnosis, prognosis, and treatment.

Endothelin-axis antagonism enhances tumor perfusion in pancreatic cancer

Delivery of therapeutic agents in pancreatic cancer (PC) is impaired due to its hypovascular and desmoplastic tumor microenvironment. The Endothelin (ET)-axis is the major regulator of vasomotor tone under physiological conditions and is highly upregulated in multiple cancers. We investigated the effect of dual endothelin receptor antagonist bosentan on perfusion and macromolecular transport in a PC cell-fibroblast co-implantation tumor model using Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI). Following bosentan treatment, the contrast enhancement ratio and wash-in rates in tumors were two- and nine times higher, respectively, compared to the controls, whereas the time to peak was significantly shorter (7.29 ± 1.29 min v/s 22.08 ± 5.88 min; p = 0.04). Importantly, these effects were tumor selective as the magnitudes of change for these parameters were much lower in muscles. Bosentan treatment also reduced desmoplasia and improved intratumoral distribution of high molecular weight FITC-dextran. Overall, these findings support that targeting the ET-axis can serve as a potential strategy to selectively enhance tumor perfusion and improve the delivery of therapeutic agents in pancreatic tumors.