Intratumoral Distribution Research Articles

Photoacoustic tracking of photo-magnetically powered nanoparticles for cancer therapy

Abstract The in vivo propulsion and monitoring of nanoparticles (NPs) have received tremendous achievements in the past decade. Developing functional NPs that can be efficiently manipulated inside the human body with a non-invasive tracking modality is critical to clinical translation. This study synthesized a photo-magnetically powered nanoparticle (PMN) with a Fe3O4 core and gold spiky surface. The Au-nanotips ensure PMNs have a strong light absorption in the second near-infrared (NIR) window and produce outstanding photoacoustic signals. The Bio-transmission electron microscopy and simulation results prove that the assembly of PMNs under a magnetic field further enhances the photothermal conversion in cells, contributing to the reduction of ambient viscosity. Photoacoustic imaging (PAI) realized real-time monitoring of PMN movements and revealed that laser plus magnetic coupling could improve intratumoral distribution and retention. The proposed methods exhibit excellent potential for the clinical research of cancer nanotherapies.

Shifting the paradigm in personalized cancer care through next-generation therapeutics and computational pathology.

The incorporation of novel therapeutic agents such as antibody-drug conjugates, radio-conjugates, T-cell engagers, and chimeric antigen receptor cell therapies represents a paradigm shift in oncology. Cell-surface target quantification, quantitative assessment of receptor internalization, and changes in the tumor microenvironment (TME) are essential variables in the development of biomarkers for patient selection and therapeutic response. Assessing these parameters requires capabilities that transcend those of traditional biomarker approaches based on immunohistochemistry, in situ hybridization and/or sequencing assays. Computational pathology is emerging as a transformative solution in this new therapeutic landscape, enabling detailed assessment of not only target presence, expression levels, and intra-tumor distribution but also of additional phenotypic features of tumor cells and their surrounding TME. Here, we delineate the pivotal role of computational pathology in enhancing the efficacy and specificity of these advanced therapeutics, underscoring the integration of novel artificial intelligence models that promise to revolutionize biomarker discovery and drug development.

Design of the distribution of iron oxide (Fe3O4) nano-particle drug in realistic cholangiocarcinoma model and the simulation of temperature increase during magnetic induction hyperthermia

The prognosis for Cholangiocarcinoma (CCA) is unfavorable, necessitating the development of new therapeutic approach such as magnetic hyperthermia therapy (MHT) which is induced by magnetic nano-particle (MNPs) drug to bridge the treatment gap. Given the deep location of CCA within the abdominal cavity and proximity to vital organs, accurately predict the individualized treatment effects and safety brought by the distribution of MNPs in tumor will be crucial for the advancement of MHT in CCA. The Mimics software was used in this study to conduct three-dimensional reconstruction of abdominal computed tomography (CT) and magnetic reso-nance imaging images from clinical patients, resulting in the generation of a realistic digital geometric model representing the human biliary tract and its adjacent structures. Subsequently, The COMSOL Multiphysics software was utilized for modeling CCA and calculating the heat transfer law resulting from the multi-regional distribution of MNPs in CCA. The temperature within the central region of irregular CCA measured approximately 46°C, and most areas within the tumor displayed temperatures surpassing 41°C. The temperature of the inner edge of CCA is only 39 ∼ 41℃, however, it can be ameliorated by adjusting the local drug concentration through simulation system. For CCA with diverse morphologies and anatomical locations, the multi-regional distribution patterns of intratumoral MNPs and a slight overlap of drug distribution areas synergistically enhance intratumoral temperature while ensuring treatment safety. The present study highlights the practicality and imperative of incorporating personalized intratumoral MNPs distribution strategy into clinical practice for MHT, which can be achieved through the development of an integrated simulation system which incorporates medical image data and numerical calculations.

Assessment of Radiolabelled Derivatives of R954 for Detection of Bradykinin B1 Receptor in Cancer Cells: Studies on Glioblastoma Xenografts in Mice.

Bradykinin B1 receptor (B1R) has garnered attention as a cancer therapeutic and diagnostic target. Several reports on radiolabelled derivatives of B1R antagonists have shown favourable properties as imaging agents in cells highly expressing hB1R following transfection. In the present study, we assessed whether radiolabelled probes can detect B1R endogenously expressed in cancer cells. To this end, we evaluated 111In-labelled derivatives of a B1R antagonist ([111In]In-DOTA-Ahx-R954) using glioblastoma cell lines (U87MG and U251MG) with different B1R expression levels. Cellular uptake studies showed that the specific accumulation of [111In]In-DOTA-Ahx-R954 in U87MG was higher than that in U251MG, which correlated with B1R expression levels. Tissue distribution in U87MG-bearing mice revealed approximately 2-fold higher radioactivity in tumours than in the muscle in the contralateral leg. The specific accumulation of [111In]In-DOTA-Ahx-R954 in the tumour was demonstrated by the reduction in the tumour-to-plasma ratios in nonlabelled R954-treated mice. Moreover, ex vivo autoradiographic images revealed that the intratumoural distribution of [111In]In-DOTA-Ahx-R954 correlated with the localisation of B1R-expressing glioblastoma cells. In conclusion, we demonstrated that [111In]In-DOTA-Ahx-R954 radioactivity correlated with B1R expression in glioblastoma cells, indicating that radiolabelled derivatives of the B1R antagonist could serve as promising tools for elucidating the involvement of B1R in cancer.

Transcriptomic response of prostate cancer cells to carbon ion and photon irradiation with focus on androgen receptor and TP53 signaling

BackgroundRadiotherapy is essential in the treatment of prostate cancer. An alternative to conventional photon radiotherapy is the application of carbon ions, which provide a superior intratumoral dose distribution and less induced damage to adjacent healthy tissue. A common characteristic of prostate cancer cells is their dependence on androgens which is exploited therapeutically by androgen deprivation therapy in the advanced prostate cancer stage. Here, we aimed to analyze the transcriptomic response of prostate cancer cells to irradiation by photons in comparison to carbon ions, focusing on DNA damage, DNA repair and androgen receptor signaling.MethodsProstate cancer cell lines LNCaP (functional TP53 and androgen receptor signaling) and DU145 (dysfunctional TP53 and androgen receptor signaling) were irradiated by photons or carbon ions and the subsequent DNA damage was assessed by immuno-cytofluorescence. Furthermore, the cells were treated with an androgen-receptor agonist. The effects of irradiation and androgen treatment on the gene regulation and the transcriptome were investigated by RT-qPCR and RNA sequencing, followed by bioinformatic analysis.ResultsFollowing photon or carbon ion irradiation, both LNCaP and DU145 cells showed a dose-dependent amount of visible DNA damage that decreased over time, indicating occurring DNA repair. In terms of gene regulation, mRNAs involved in the TP53-dependent DNA damage response were significantly upregulated by photons and carbon ions in LNCaP but not in DU145 cells, which generally showed low levels of gene regulation after irradiation. Both LNCaP and DU145 cells responded to photons and carbon ions by downregulation of genes involved in DNA repair and cell cycle, partially resembling the transcriptome response to the applied androgen receptor agonist. Neither photons nor carbon ions significantly affected canonical androgen receptor-dependent gene regulation. Furthermore, certain genes that were specifically regulated by either photon or carbon ion irradiation were identified.ConclusionPhoton and carbon ion irradiation showed a significant congruence in terms of induced signaling pathways and transcriptomic responses. These responses were strongly impacted by the TP53 status. Nevertheless, irradiation mode-dependent distinct gene regulations with undefined implication for radiotherapy outcome were revealed. Androgen receptor signaling and irradiations shared regulation of certain genes with respect to DNA-repair and cell-cycle.

Optimizing Solid Tumor Treatment with Antibody-drug Conjugates Using Agent-Based Modeling: Considering the Role of a Carrier Dose and Payload Class.

Antibody-drug conjugates (ADCs) show significant clinical efficacy in the treatment of solid tumors, but a major limitation to their success is poor intratumoral distribution. Adding a carrier dose improves both distribution and overall drug efficacy of ADCs, but the optimal carrier dose has not been outlined for different payload classes. In this work, we study two carrier dose regimens: 1) matching payload potency to cellular delivery but potentially not reaching cells farther away from blood vessels, or 2) dosing to tumor saturation but risking a reduction in cell killing from a lower amount of payload delivered per cell. We use a validated computational model to test four different payloads conjugated to trastuzumab to determine the optimal carrier dose as a function of target expression, ADC dose, and payload potency. We find that dosing to tumor saturation is more efficacious than matching payload potency to cellular delivery for all payloads because the increase in thenumber of cells targeted by the ADC outweighs the loss in cell killing on targeted cells. An important exception exists if the carrier dose reduces the payload uptake per cell to the point where all cell killing is lost. Likewise, receptor downregulation can mitigate the benefits of a carrier dose. Because tumor saturation and in vitro potency can be measured early in ADC design, these results provide insight into maximizing ADC efficacy and demonstrate the benefits of using simulation to guide ADC design.

Pharmacokinetic-Pharmacodynamic Analysis of pH-Responsive Doxorubicin-Releasing Micelles with Anticancer Activity.

This study aimed to investigate the effect of in vivo pH-responsive doxorubicin (DOX) release and the targetability of pilot molecules in folic acid (FA)-modified micelles using a pharmacokinetic-pharmacodynamic (PK-PD) model. The time profiles of intratumoral DOX concentrations in Walker256 tumor-bearing rats were monitored using a microdialysis probe, followed by compartmental analysis, to evaluate intratumoral tissue pharmacokinetics. Maximal DOX was released from micelles 350 min after the administration of pH-responsive DOX-releasing micelles. However, FA modification of the micelles shortened the time to peak drug concentration to 150 min. Additionally, FA modification resulted in a 27-fold increase in the tumor inflow rate constant. Walker256 tumor-bearing rats were subsequently treated with DOX, pH-responsive DOX-releasing micelles, and pH-responsive DOX-releasing FA-modified micelles to monitor the tumor growth-time profiles. An intratumoral threshold concentration of DOX (55-64 ng/g tumor) was introduced into the drug efficacy compartment to construct a PD model, followed by PK-PD analysis of the tumor growth-time profiles. Similar results of threshold concentration and drug potency of DOX were obtained across all three formulations. Cell proliferation was delayed as the drug delivery ability of DOX was improved. The PK model, which was developed using the microdialysis method, revealed the intratumoral pH-responsive DOX distribution profiles. This facilitated the estimation of intratumoral PK parameters. The PD model with threshold concentrations contributed to the estimation of PD parameters in the three formulations, with consistent mechanisms observed. We believe that our PK-PD model can objectively assess the contributions of pH-responsive release ability and pilot molecule targetability to pharmacological effects.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Suppressing Wnt signaling of the blood‒tumor barrier to intensify drug delivery and inhibit lipogenesis of brain metastases

Lipogenesis is often highly upregulated in breast cancer brain metastases to adapt to intracranial low lipid microenvironments. Lipase inhibitors hold therapeutic potential but their intra-tumoral distribution is often blocked by the blood‒tumor barrier (BTB). BTB activates its Wnt signaling to maintain barrier properties, e.g., Mfsd2a-mediated BTB low transcytosis. Here, we reported VCAM-1-targeting nano-wogonin (W@V-NPs) as an adjuvant of nano-orlistat (O@V-NPs) to intensify drug delivery and inhibit lipogenesis of brain metastases. W@V-NPs were proven to be able to inactivate BTB Wnt signaling, downregulate BTB Mfsd2a, accelerate BTB vesicular transport, and enhance tumor accumulation of O@V-NPs. With the ability to specifically kill cancer cells in a lipid-deprived environment with IC50 at 48 ng/mL, W@V-NPs plus O@V-NPs inhibited the progression of brain metastases with prolonged survival of model mice. The combination did not induce brain edema, cognitive impairment, and systemic toxicity in healthy mice. Targeting Wnt signaling could safely modulate the BTB to improve drug delivery and metabolic therapy against brain metastases.

Biopolymer-based tumor microenvironment-responsive nanomedicine for targeted cancer therapy.

Nanomedicine has opened up new avenues for cancer treatment by enhancing drug solubility, permeability and targeted delivery to cancer cells. Despite its numerous advantages over conventional therapies, nanomedicine may exhibit off-target drug distribution, harming nontarget regions. The increased permeation and retention effect of nanomedicine in tumor sites also has its limitations, as abnormal tumor vasculature, dense stroma structure and altered tumor microenvironment (TME) may result in limited intratumor distribution and therapeutic failure. However, TME-responsive nanomedicine has exhibited immense potential for efficient, safe and precise delivery of therapeutics utilizing stimuli specific to the TME. This review discusses the mechanistic aspects of various TME-responsive biopolymers and their application in developing various types of TME-responsive nanomedicine.

Radionuclide Reporter Imaging to Visualize Tumor Hypoxia Ex Vivo and In Vivo.

In vitro studies using cell culture, including three-dimensional cultures without the involvement of tumor vessels, have limitations in simulating complex intratumoral hypoxic conditions in live subjects. To generate experimental hypoxic conditions closer to those observed in humans in clinical settings, in vivo studies are necessary. In addition, visible light generated via bioluminescence and fluorescence is generally unsuitable for in vivo experiments because of low tissue penetration. Furthermore, near-infrared light (NIR), which has the highest tissue penetration among lights of different wavelengths, cannot be assessed precisely in vivo because of the difficulty in correcting tissue absorption and scatter. For in vivo quantitative analyses, imaging modalities that use high tissue-penetrating signals, such as computed tomography (CT) using X-rays, radionuclide imaging using γ-rays, and magnetic resonance imaging (MRI) using electromagnetic waves, are ideal.Therefore, as an advanced protocol for this research purpose, we provide ex vivo and in vivo methods to investigate the genetic response of multiple copies of hypoxia response elements (HREs) to tumor hypoxia in terms of intensity and intratumoral distribution using a human sodium/iodide symporter (hNIS) reporter gene and radionuclide reporter probes (radioiodine and its chemical analog Tc-99m) based on our previous research. This protocol includes cloning an hNIS reporter construct with multiple copies of HREs, establishing stable cell lines of the reporter construct, preparing a mouse subcutaneous xenograft model, and evaluating the genetic response of multiple HREs to tumor hypoxia using digital autoradiography (ARG) ex vivo and using single-photon emission computed tomography (SPECT) or positron emission tomography (PET) in vivo.

Ru(dpp)3]Cl2-Embedded Oxygen Nano Polymeric Sensors: A Promising Tool for Monitoring Intracellular and Intratumoral Oxygen Gradients with High Quantum Yield and Long Lifetime.

Unraveling the intricacies between oxygen dynamics and cellular processes in the tumor microenvironment (TME) hinges upon precise monitoring of intracellular and intratumoral oxygen levels, which holds paramount significance. The majority of these reported oxygen nanoprobes suffer compromised lifetime and quantum yield when exposed to the robust ROS activities prevalent in TME, limiting their prolonged in vitro usability. Herein, the ruthenium-embedded oxygen nano polymeric sensor (Ru-ONPS) is proposed for precise oxygen gradient monitoring within the cellular environment and TME. Ru-ONPS (≈64±7nm) incorporates [Ru(dpp)3]Cl2 dye into F-127 and crosslinks it with urea and paraformaldehyde, ensuring a prolonged lifetime (5.4 µs), high quantum yield (66.65±2.43% in N2 and 49.80±3.14% in O2), superior photostability (>30 min), and excellent stability in diverse environmental conditions. Based on the Stern-Volmer plot, the Ru-ONPS shows complete linearity for a wide dynamic range (0-23 mg L-1), with a detection limit of 10 µg mL-1. Confocal imaging reveals Ru-ONPS cellular uptake and intratumoral distribution. After 72 h, HCT-8 cells show 5.20±1.03% oxygen levels, while NIH3T3 cells have 7.07±1.90%. Co-culture spheroids display declining oxygen levels of 17.90±0.88%, 10.90±0.88%, and 5.10±1.18%, at 48, 120, and 216 h, respectively. Ru-ONPS advances cellular oxygen measurement and facilitates hypoxia-dependent metastatic research and therapeutic target identification.

Development and Utility of an Imaging System for Internal Dosimetry of Astatine-211 in Mice.

In targeted radionuclide therapy, determining the absorbed dose of the ligand distributed to the whole body is vital due to its direct influence on therapeutic and adverse effects. However, many targeted alpha therapy drugs present challenges for in vivo quantitative imaging. To address this issue, we developed a planar imaging system equipped with a cadmium telluride semiconductor detector that offers high energy resolution. This system also comprised a 3D-printed tungsten collimator optimized for high sensitivity to astatine-211, an alpha-emitting radionuclide, and adequate spatial resolution for mouse imaging. The imager revealed a spectrum with a distinct peak for X-rays from astatine-211 owing to the high energy resolution, clearly distinguishing these X-rays from the fluorescent X-rays of tungsten. High collimator efficiency (4.5 × 10-4) was achieved, with the maintenance of the spatial resolution required for discerning mouse tissues. Using this system, the activity of astatine-211 in thyroid cancer tumors with and without the expression of the sodium iodide symporter (K1-NIS/K1, respectively) was evaluated through in vivo imaging. The K1-NIS tumors had significantly higher astatine-211 activity (sign test, p = 0.031, n = 6) and significantly decreased post-treatment tumor volume (Student's t-test, p = 0.005, n = 6). The concurrent examination of intratumor drug distribution and treatment outcome could be performed with the same mice.

Bench to Bedside Development of [18F]Fluoromethyl-(1,2-2H4)choline ([18F]D4-FCH).

Malignant transformation is characterised by aberrant phospholipid metabolism of cancers, associated with the upregulation of choline kinase alpha (CHKα). Due to the metabolic instability of choline radiotracers and the increasing use of late-imaging protocols, we developed a more stable choline radiotracer, [18F]fluoromethyl-[1,2-2H4]choline ([18F]D4-FCH). [18F]D4-FCH has improved protection against choline oxidase, the key choline catabolic enzyme, via a 1H/2D isotope effect, together with fluorine substitution. Due to the promising mechanistic and safety profiles of [18F]D4-FCH in vitro and preclinically, the radiotracer has transitioned to clinical development. [18F]D4-FCH is a safe positron emission tomography (PET) tracer, with a favourable radiation dosimetry profile for clinical imaging. [18F]D4-FCH PET/CT in lung and prostate cancers has shown highly heterogeneous intratumoral distribution and large lesion variability. Treatment with abiraterone or enzalutamide in metastatic castrate-resistant prostate cancer patients elicited mixed responses on PET at 12-16 weeks despite predominantly stable radiological appearances. The sum of the weighted tumour-to-background ratios (TBRs-wsum) was associated with the duration of survival.

A High‐Resolution Prediction Network for Predicting Intratumoral Distribution of Nanoprobes by Tumor Vascular and Nuclear Feature

In this study, the critical need for precise and accurate prediction of intra‐tumor heterogeneity related to the enhanced permeability and retention effect and spatial distribution of nanoprobes is addressed for the development of effective nanodrug delivery strategies. Current predictive models are limited in terms of resolution and accuracy, prompting the construction of a high‐resolution prediction network (HRPN) that estimates the microdistribution of quantum dots, factoring in tumor vascular and nuclear features. The HRPN algorithm is trained using 27 780 patches and validated on 4920 patches derived from 4T1 breast cancer whole‐slide images, demonstrating its reliability. The HRPN model exhibits minimal error (mean square error = 1.434, root mean square error = 1.198), satisfactory goodness of fit (R2 = 0.891), and superior image quality (peak signal‐to‐noise ratio = 44.548) when compared to a generative‐adversarial‐network‐structured model. Furthermore, the HRPN model offers improved prediction accuracy, broader prediction intervals, and reduced computational resource requirements. Consequently, the proposed model yields high‐resolution predictions that more closely resemble actual tumor microdistributions, potentially serving as a powerful analytical tool for investigating the spatial relationship between the tumor microenvironment and nanoprobes.

TMIC-56. DECIPHERING THE INTRICATE GLIOBLASTOMA TUMOR MICROENVIRONMENT AND EGFRVIII TRANSCRIPT DISTRIBUTION THROUGH SINGLE-CELL SPATIAL PROFILING

Abstract INTRODUCTION Profiling the tumor microenvironment (TME) in glioblastoma (GBM) poses challenges due to its complex and heterogeneous nature. Analyzing diverse cell populations, deciphering dynamic cell-cell interactions, and overcoming spatial-temporal variations present significant challenges to characterizing the GBM TME. These challenges can, in part, be overcome using in situ sequencing (ISS) approaches that offer the advantage of directly analyzing gene expression within intact tissue, providing spatial and molecular context for understanding complex biological processes and tumor heterogeneity. Method/ RESULTS Here, we report a ‘first in world’ application of 10xGenomics Xenium ISS platform to profiling RNA expression profiles within GBM tissue, including primary and recurrent tumors, to establish single cell-level profiling of the TME. Our panel of canonical and custom GBM genes allowed us to establish profiles of high-resolution cell neighborhoods, intratumoral cell-types and cell-states, and the distribution of infiltrating tumor cells throughout the tissue. Exploiting the chemistry that underlies the Xenium platform, we also developed a novel ISS isoform expression assay that semi-quantitatively profiles a mutated variant of the epidermal growth factor receptor (EGFR). Data show that EGFRvIII, which has been shown to promote tumor growth, invasion, and resistance to therapies, is preferentially expressed within tumor-subsets within the GBM. Our EGFRvIII assay not only has the potential to act as a high sensitivity method of tumor diagnosis but its distribution will allow for correlation of this tumorigenic marker with activated pathways across the tissue. CONCLUSION ISS is a valuable tool for profiling the complex TME in GBM, enabling analysis of intratumoral cell types, cell states, and EGFRvIII distribution. Our study demonstrates its pioneering application, offering insights into the molecular landscape and potential for novel diagnostic signatures and improved treatment strategies for this aggressive cancer.

EPCO-09. CHARACTERIZING THE GBM CELLULAR LANDSCAPE BY LARGE-SCALE SINGLE-NUCLEUS RNA-SEQUENCING

Abstract Tumor heterogeneity is a well-known hallmark of glioblastoma (GBM). Single-cell RNA-sequencing (scRNA-seq) technologies significantly extended our understanding of GBM intra-tumor heterogeneity. While the landmark scRNA-seq studies were limited by scale, recent technological advances have enabled analyzing increasing numbers of cells. Here, leveraging a large-scale single-nucleus RNA-sequencing (snRNA-seq) dataset containing 457,442 cells (271,444 malignant and 185,995 non-malignant) from 121 IDH-wildtype GBM tumor samples and matching whole-exome/whole-genome sequencing data, we dissected the GBM malignant and tumor microenvironment (TME) compartments with unprecedented resolution and scale. We identified three novel malignant cellular states - Glial progenitor cell-like (GPC-like), Neuron-like and Cilia-like - in addition to the cellular states previously defined in GBM (NPC-like, OPC-like, AC-like and MES-like). Cross-referencing our dataset with published scRNA-seq datasets suggests that GBM states mirror the developmental hierarchy observed in the developing human brain. Functional enrichment analysis demonstrated the heterogeneous nature of pathway and metabolic activities across the spectrum of cellular states. Analysis of the matched DNA-sequencing data revealed novel associations between the occurrence of certain genetic events and the abundance of specific cellular states. Similarly, we dissected the TME and robustly defined cellular states for different TME cell types, underscoring the heterogeneity found also within the TME compartment. Leveraging the large number of samples in our dataset and controlling for intra-tumor cell state frequency exposed three baseline gene expression programs - neuronal activity, glial development and extra-cellular matrix remodeling - termed State-Controlled Profiles (SCPs), that reflect the inter-tumor heterogeneity and are not influenced by the intra-tumor cell state distribution. Integrated analysis of the intra- and inter-tumor heterogeneity components revealed striking associations between certain TME cell types and states, malignant cell states and SCPs, suggesting that an interplay between the TME and malignant cells shapes the frequency of malignant states and baseline expression profile in each tumor.

Assessment of Brain Tumour Perfusion Using Early-Phase 18F-FET PET: Comparison with Perfusion-Weighted MRI

PurposeMorphological imaging using MRI is essential for brain tumour diagnostics. Dynamic susceptibility contrast (DSC) perfusion-weighted MRI (PWI), as well as amino acid PET, may provide additional information in ambiguous cases. Since PWI is often unavailable in patients referred for amino acid PET, we explored whether maps of relative cerebral blood volume (rCBV) in brain tumours can be extracted from the early phase of PET using O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET).ProcedureUsing a hybrid brain PET/MRI scanner, PWI and dynamic 18F-FET PET were performed in 33 patients with cerebral glioma and four patients with highly vascularized meningioma. The time interval from 0 to 2 min p.i. was selected to best reflect the blood pool phase in 18F-FET PET. For each patient, maps of MR-rCBV, early 18F-FET PET (0–2 min p.i.) and late 18F-FET PET (20–40 min p.i.) were generated and coregistered. Volumes of interest were placed on the tumour (VOI-TU) and normal-appearing brain (VOI-REF). The correlation between tumour-to-brain ratios (TBR) of the different parameters was analysed. In addition, three independent observers evaluated MR-rCBV and early 18F-FET maps (18F-FET-rCBV) for concordance in signal intensity, tumour extent and intratumoural distribution.ResultsTBRs calculated from MR-rCBV and 18F-FET-rCBV showed a significant correlation (r = 0.89, p < 0.001), while there was no correlation between late 18F-FET PET and MR-rCBV (r = 0.24, p = 0.16) and 18F-FET-rCBV (r = 0.27, p = 0.11). Visual rating yielded widely agreeing findings or only minor differences between MR-rCBV maps and 18F-FET-rCBV maps in 93 % of the tumours (range of three independent raters 91–94%, kappa among raters 0.78–1.0).ConclusionEarly 18F-FET maps (0–2 min p.i.) in gliomas provide similar information to MR-rCBV maps and may be helpful when PWI is not possible or available. Further studies in gliomas are needed to evaluate whether 18F-FET-rCBV provides the same clinical information as MR-rCBV.

Integrated PK/PD Modeling Relates Smoothened Inhibitor Biomarkers to The Heterogeneous Intratumor Disposition of Cetuximab in Pancreatic Cancer Tumor Models

Therapeutic antibodies have shown little efficacy in the treatment of pancreatic ductal adenocarcinomas (PDAC). Tumor desmoplasia, hypovascularity, and poor perfusion result in insufficient tumor cell exposure, contributing to treatment failure. Smoothened inhibitors of hedgehog signaling (sHHi) increase PDAC tumor permeability, perfusion, and drug delivery, and provide a tool to develop a quantitative, mechanistic understanding as to how the temporal dynamics of tumor priming can impact intratumor distribution of monoclonal antibodies (mAb). A linked pharmacokinetic (PK)/pharmacodynamic (PD) model was developed to integrate the plasma and tumor PK of a sHHi priming agent with its effects upon downstream stromal biomarkers Gli1, hyaluronic acid, and interstitial fluid pressure in PDAC patient-derived xenograft (PDX) tumors. In parallel, in situ tumor concentrations of cetuximab (CTX: anti-epidermal growth factor receptor; EGFR) were quantified as a marker for tumor delivery of mAb or antibody-drug conjugates. A minimal, physiologically-based pharmacokinetic (mPBPK) model was constructed to link sHHi effects upon mechanistic effectors of tumor barrier compromise with the intratumor distribution of CTX, and CTX occupancy of EGFR in tumors. Integration of the mPBPK model of mAb deposition and intratumor distribution with the PK/PD model of tumor responses to priming not only identified physiological parameters that are critical for tumor antibody distribution, but also provides insight into dosing regimens that could achieve maximal tumor disposition of therapeutic antibodies under conditions of transient PDAC tumor permeability barrier compromise that mechanistically-diverse tumor priming strategies may achieve.

Head-to-head comparison of three chelates reveals DOTAGA promising for 225 Ac labeling of anti-FZD10 antibody OTSA101.

To select the most suitable chelate for 225 Ac radiolabeling of the anti-FZD10 antibody OTSA101, we directly compared three chelates: S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2,2',2″-(10-(1-carboxy-4-((4-isothiocyanatobenzyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (p-SCN-Bn-DOTAGA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DO3A-NHS-ester). We evaluated the binding affinity of the chelate-conjugated OTSA101 antibodies, as well as the labeling efficiency and stability in murine serum of 225 Ac-labeled OTSA101 as in vitro properties. The biodistribution, intratumoral distribution, absorbed doses, and therapeutic effects of the chelate-conjugated OTSA101 antibodies were assessed in the synovial sarcoma mouse model SYO-1. Of the three conjugates, DOTAGA conjugation had the smallest impact on the binding affinity (p < 0.01). The labeling efficiencies of DOTAGA-OTSA101 and DO3A-OTSA101 were 1.8-fold higher than that of DOTA-OTSA101 (p < 0.01). The stabilities were similar between 225 Ac-labeled DOTA-OTSA101, DOTAGA-OTSA101, and DO3A-OTSA101in serum at 37 and 4°C. The dosimetric analysis based on the biodistribution revealed significantly higher tumor-absorbed doses by 225 Ac-labeled DOTA-OTSA101 and DOTAGA-OTSA101 compared with 225 Ac-DO3A-OTSA101 (p < 0.05). 225 Ac-DOTAGA-OTSA101 exhibited the highest tumor-to-bone marrow ratio, with bone marrow being the dose-limiting tissue. The therapeutic and adverse effects were not significantly different between the three conjugates. Our findings indicate that among the three evaluated chelates, DOTAGA appears to be the most promising chelate to produce 225 Ac-labeled OTSA101 with high binding affinity and high radiochemical yields while providing high absorbed doses to tumors and limited absorbed doses to bone marrow.