Intratumoral Distribution Research Articles

MEDB-66. Investigating intra-tumoral heterogeneity of extrachromosomal DNA in SHH medulloblastoma

Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. Our study’s aim is to better understand how ecDNA containing cells can potentiate malignant growth. EcDNA’s role in the development of treatment resistance and association with poor outcomes is hypothesized to arise from its contribution to intra-tumoral heterogeneity and its potential to promote oncogene dependency switching. To analyze the intra-tumoral distribution of ecDNA, we have now simultaneously analyzed the accessible chromatin and gene expression in single cells of a SHH medulloblastoma (MB) patient using multiome single-cell ATAC-seq and gene expression (10X Genomics). Whole genome sequencing (WGS) of this tumor previously revealed a heterozygous somatic TP53 mutation and two distinct ecDNAs: a 3.2Mbp amplicon comprising 3 regions of chr1 and another 4.5Mbp amplicon comprising 23 segments originating from chr7 and chr17. We then used multimodal analysis to describe the tumor cell types, gene expression, variant signatures and estimate ecDNA copy number in the medulloblastoma tumor sample. We identified 12 distinct clusters in the human tumor, 5 of which were determined to be normal non-tumor [OSC1] cells, as identified by specific cell type markers, and 7 of which were determined to be tumor cells. Enrichment of ecDNA was restricted to only one of these tumor clusters. In addition, we also performed the same multiome single-cell analyses in an orthotopic xenograft mouse model derived from this SHH MB patient tumor. In the PDX, 17 clusters were identified, all of which were determined to be tumor cells and enriched for ecDNA. Our preliminary results indicate that tumor cells with ecDNA in the human tumor (particularly the ecDNA enriched cluster) almost exclusively account for [OSC2] the cells in the corresponding PDX, emphasizing the aggressiveness of ecDNA containing cells.

Changeable net charge on nanoparticles facilitates intratumor accumulation and penetration.

The Enhanced Permeability and Retention (EPR) effect is a golden strategy for the nanoparticle (NP)-based targeting of solid tumors, and the surface property of NPs might be a determinant on their targeting efficiency. Poly(ethylene glycol) (PEG) is commonly used as a shell material; however, it has been pointed out that PEG-coated NPs may exhibit accumulation near tumor vasculature rather than having homogenous intratumor distribution. The PEG shell plays a pivotal role on prolonged blood circulation of NPs but potentially impairs the intratumor retention of NPs. In this study, we report on a shell material to enhance tumor-targeted delivery of NPs by maximizing the EPR effect: polyzwitterion based on ethylenediamine-based carboxybetaine [PGlu(DET-Car)], which shows the changeable net charge responding to surrounding pH. The net charge of PGlu(DET-Car), is neutral at physiological pH7.4, allowing it to exhibit a stealth property during the blood circulation; however, it becomes cationic for tissue-interactive performance under tumorous acidic conditions owing to the stepwise protonation behavior of ethylenediamine. Indeed, the PGlu(DET-Car)-coated NPs (i.e., gold NPs in the present study) exhibited prolonged blood circulation and remarkably enhanced tumor accumulation and retention than PEG-coated NPs, achieving 32.1% of injected dose/g of tissue, which was 4.2 times larger relative to PEG-coated NPs. Interestingly, a considerable portion of PGlu(DET-Car)-coated NPs clearly penetrated into deeper tumor sites and realized the effective accumulation in hypoxic regions, probably because the cationic net charge of PGlu(DET-Car) is augmented in more acidic hypoxic regions. This study suggests that the changeable net charge on the NP surface in response to tumorous acidic conditions is a promising strategy for tumor-targeted delivery based on the EPR effect.

New Cluster Analysis Method for Quantitative Dynamic Contrast-Enhanced MRI Assessing Tumor Heterogeneity Induced by a Tumor-Microenvironmental Ameliorator (E7130) Treatment to a Breast Cancer Mouse Model.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide insight into tumor perfusion. However, a method that can quantitatively measure the intra-tumor distribution of tumor voxel clusters with a distinct range of Ktrans and ve values remains insufficiently explored. Two-dimensional cluster analysis may quantify the distribution of a tumor voxel subregion with a distinct range of Ktrans and ve values in human breast cancer xenografts. Prospective longitudinal study. Twenty-two female athymic nude mice with MCF-7 xenograft, treated with E7130, a tumor-microenvironmental ameliorator, or saline. 9.4 Tesla, turbo rapid acquisition with relaxation enhancement, and spoiled gradient-echo sequences. We performed two-dimensional k-means clustering to identify tumor voxel clusters with a distinct range of Ktrans and ve values on Days 0, 2, and 5 after treatment, calculated the ratio of the number of tumor voxels in each cluster to the total number of tumor voxels, and measured the normalized distances defined as the ratio of the distance between each tumor voxel and the nearest tumor margin to a tumor radius. Unpaired t-tests, Dunnett's multiple comparison tests, and Chi-squared test were used. The largest and second largest clusters constituted 44.4% and 27.5% of all tumor voxels with cluster centroid values of Ktrans at 0.040 min-1 and 0.116 min-1 , and ve at 0.131 and 0.201, respectively. At baseline (Day 0), the average normalized distances for the largest and second largest clusters were 0.33 and 0.24, respectively. E7130-treated group showed the normalized distance of the initial largest cluster decreasing to 0.25, while that of the second largest cluster increasing to 0.31. Saline-treated group showed no change. A two-dimensional cluster analysis might quantify the spatial distribution of a tumor subregion with a distinct range of Ktrans and ve values. 1 TECHNICAL EFFICACY: Stage 1.

Factors for Differential Outcome Across Cancers in Clinical Molecule-Targeted Fluorescence Imaging.

Clinical imaging performance using a fluorescent antibody was compared across 3 cancers to elucidate physical and biologic factors contributing to differential translation of epidermal growth factor receptor (EGFR) expression to macroscopic fluorescence in tumors. Methods: Thirty-one patients with high-grade glioma (HGG, n = 5), head-and-neck squamous cell carcinoma (HNSCC, n = 23), or lung adenocarcinoma (LAC, n = 3) were systemically infused with 50 mg of panitumumab-IRDye800 1-3 d before surgery. Intraoperative open-field fluorescent images of the surgical field were acquired, with imaging device settings and operating room lighting conditions being tested on tissue-mimicking phantoms. Fluorescence contrast and margin size were measured on resected specimen surfaces. Antibody distribution and EGFR immunoreactivity were characterized in macroscopic and microscopic histologic structures. The integrity of the blood-brain barrier was examined via tight junction protein (Claudin-5) expression with immunohistochemistry. Stepwise multivariate linear regression of biologic variables was performed to identify independent predictors of panitumumab-IRDye800 concentration in tissue. Results: Optimally acquired at the lowest gain for tumor detection with ambient light, intraoperative fluorescence imaging enhanced tissue-size dependent tumor contrast by 5.2-fold, 3.4-fold, and 1.4-fold in HGG, HNSCC, and LAC, respectively. Tissue surface fluorescence target-to-background ratio correlated with margin size and identified 78%-97% of at-risk resection margins ex vivo. In 4-μm-thick tissue sections, fluorescence detected tumor with 0.85-0.89 areas under the receiver-operating-characteristic curves. Preferential breakdown of blood-brain barrier in HGG improved tumor specificity of intratumoral antibody distribution relative to that of EGFR (96% vs. 80%) despite its reduced concentration (3.9 ng/mg of tissue) compared with HNSCC (8.1 ng/mg) and LAC (6.3 ng/mg). Cellular EGFR expression, tumor cell density, plasma antibody concentration, and delivery barrier were independently associated with local intratumoral panitumumab-IRDye800 concentration, with 0.62 goodness of fit of prediction. Conclusion: In multicancer clinical imaging of a receptor-ligand-based molecular probe, plasma antibody concentration, delivery barrier, and intratumoral EGFR expression driven by cellular biomarker expression and tumor cell density led to heterogeneous intratumoral antibody accumulation and spatial distribution whereas tumor size, resection margin, and intraoperative imaging settings substantially influenced macroscopic tumor contrast.

Appropriate Size of Fe3O4 Nanoparticles for Cancer Therapy by Ferroptosis.

Iron oxide nanoparticles can induce cell death due to the ferroptosis mechanism, showing a great potential for cancer therapy. Here, we synthesized different-sized iron oxide nanoparticles (2-100 nm) to investigate their antitumor effect and toxicity mechanism. It was found that ultrasmall nanoparticles (< ∼5 nm) could accumulate in nucleus and were more efficient in triggering the generation of •OH than larger nanoparticles due to the quicker release of Fe2+, thus exhibiting more remarkable cytotoxicity. Nevertheless, 10 nm iron oxide nanoparticles group displayed the best antitumor effect in vivo. We studied the in vivo and intratumoral biodistribution of the nanoparticles and found that the therapeutic effects were related to both the tumoral accumulation and intratumoral distribution of nanoparticles. This work indicates the appropriate size of Fe3O4 NPs for cancer treatment and illustrates the possible factors that influence the therapeutic effect, suggesting the great potential of iron oxide in clinical application.

Engineering biomolecular systems: Controlling the self-assembly of gelatin to form ultra-small bioactive nanomaterials.

The size of nanocarriers determines the biological property of the materials, especially as it relates to intratumoral distribution. Previous research has shown that sizes of 10–50 nm penetrate deep inside the tumor, resulting in better efficacy. On the other hand, studies have shown that gelatin exhibits excellent biological properties, including compatibility, degradability, and toxicity. Therefore, FDA approved gelatin as a safe material to use as an excipient in injectables. The bottleneck is the nonexistence of smaller-sized gelatin nanoparticles (GNPs) to realize the full potential of these biomaterials. Yet, GNPs with sizes of less than 50 nm have not been reported; the synthetic strategy reported in the literature uses “uncontrolled crosslinking coupled with nanoprecipitation”, resulting in larger particle size. We have developed a new method to self-assemble gelatin strands by using an anionic, phosphate-based crosslinker and controlled precipitation. The method we developed produced ultra-small gelatin nanoparticles (GX) of size 10 nm with a high degree of reproducibility, and it was characterized using dynamic light scattering (DLS), Energy-dispersive X-ray spectroscopy (EDS), High-resolution transmission, and scanning electron microscopy (HR-TEM/STEM). We also explored GX as a bioactive platform to encapsulate imaging and therapy agents within the cavity. Interestingly, we were able to encapsulate 2 nm size gold nanoparticles within the void of GX. The versatile nature of the GX particles was further demonstrated by surface functionalizing with larger size gelatin nanoparticles to form core-satellite nanocomposites. Additionally, we studied the tumor penetrability of dye-tagged 10, 50, and 200 nm gelatin nanoparticles. The study showed that smaller size gelatin nanoparticles penetrate deeper tumor regions than larger particles. In general, GX was efficient in penetrating the inner region of the spheroids. The results demonstrate the potential capabilities of ultra-small GX nanoparticles for multi-staged payload delivery, diagnostics, and cancer therapy.

Inducing Biomechanical Heterogeneity in Brain Tumor Modeling by MR Elastography: Effects on Tumor Growth, Vascular Density and Delivery of Therapeutics.

Simple SummaryBiomechanical forces aggravate brain tumor progression. In this study, magnetic resonance elastography (MRE) is employed to extract tissue biomechanical properties from five glioblastoma patients and a healthy subject, and data are incorporated in a mathematical model that simulates tumor growth. Mathematical modeling enables further understanding of glioblastoma development and allows patient-specific predictions for tumor vascularity and delivery of drugs. Incorporating MRE data results in a more realistic intratumoral distribution of mechanical stress and anisotropic tumor growth and a better description of subsequent events that are closely related to the development of stresses, including heterogeneity of the tumor vasculature and intrapatient variations in tumor perfusion and delivery of drugs.The purpose of this study is to develop a methodology that incorporates a more accurate assessment of tissue mechanical properties compared to current mathematical modeling by use of biomechanical data from magnetic resonance elastography. The elastography data were derived from five glioblastoma patients and a healthy subject and used in a model that simulates tumor growth, vascular changes due to mechanical stresses and delivery of therapeutic agents. The model investigates the effect of tumor-specific biomechanical properties on tumor anisotropic growth, vascular density heterogeneity and chemotherapy delivery. The results showed that including elastography data provides a more realistic distribution of the mechanical stresses in the tumor and induces anisotropic tumor growth. Solid stress distribution differs among patients, which, in turn, induces a distinct functional vascular density distribution—owing to the compression of tumor vessels—and intratumoral drug distribution for each patient. In conclusion, incorporating elastography data results in a more accurate calculation of intratumoral mechanical stresses and enables a better mathematical description of subsequent events, such as the heterogeneous development of the tumor vasculature and intrapatient variations in tumor perfusion and delivery of drugs.

Intratumoural spatial distribution of S100B\u2009+\u2009folliculostellate cells is associated with proliferation and expression of FSH and ER\u03b1 in gonadotroph tumours

Folliculostellate cells are S100B-expressing cells with numerous functions in the normal anterior pituitary. These cells have also been identified in pituitary neuroendocrine tumours (PitNETs), where their precise role remains elusive. Here, we aimed to build a refined cartography of S100B-expressing cells to characterise their interpatient and intratumoural spatial distribution, and to start identifying their potential functions in PitNETs. High-throughput histological analysis of S100B-stained tumour sections of 54 PitNETs revealed a significant decrease in S100B + cells in PitNETs compared to the normal anterior pituitary. A Ki67 index ≥ 3, a mitosis count > 2/10 per high power fields, and a proliferative status, were all associated with fewer S100B + cells in gonadotroph tumours. Gonadotroph tumours also showed interpatient and intratumoural heterogeneity in the spatial distribution of S100B + cells. The existence of an intratumoural heterogeneity was further confirmed by the incorporation to our spatial analysis of additional markers: Ki67, FSH, LH, ERα and SSTR2. The tumour areas with fewer S100B + cells displayed a higher percentage of Ki67 + cells, whereas strong positive correlations were observed between S100B + , FSH + , and ERα + cells. Such spatial associations suggest that S100B + folliculostellate cells could play a role in gonadotroph tumorigenesis, and may contribute to the maintenance of tumour cells in a low proliferating, FSH + /ERα + differentiated state. Albeit, further in-depth functional studies are required to decipher the mechanisms underlying these spatial associations and to potentially identify a therapeutic use.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents

BackgroundWhile immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics.ResultsTo introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy.ConclusionsA set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.Graphical

Comparative Study on the Efficacy and Exposure of Molecular Target Agents in Non-small Cell Lung Cancer PDX Models with Driver Genetic Alterations.

Patient-derived xenografts (PDX) can adequately reflect clinical drug efficacy. However, the methods for evaluating drug efficacy are not fully established. We selected five non-small cell lung cancer (NSCLC) PDXs with genetic alterations from established PDXs and the corresponding molecular targeted therapy was administered orally for 21 consecutive days. Genetic analysis, measurement of drug concentrations in blood and tumors using LC/MS-MS, and analysis of drug distribution in tumors using matrix-assisted laser desorption/ionization mass spectrometry were performed. Fifteen (20%) PDXs were established using samples collected from 76 patients with NSCLC with genetic alterations. The genetic alterations observed in original patients were largely maintained in PDXs. We compared the drug efficacy in original patients and PDX models; the efficacies against certain PDXs correlated with the clinical effects, while those against the others did not. We determined blood and intratumor concentrations in the PDX model, but both concentrations were low, and no evident correlation with the drug efficacy could be observed. The intratumoral spatial distribution of the drugs was both homogeneous and heterogeneous for each drug, and the distribution was independent of the expression of the target protein. The evaluation of drug efficacy in PDXs enabled partial reproduction of the therapeutic effect in original patients. A more detailed analysis of systemic and intratumoral pharmacokinetics may help clarify the mode of action of drugs. Further development of evaluation methods and indices to improve the prediction accuracy of clinical efficacy is warranted.

DNA-assembled visible nanodandelions with explosive hydrogen-bond breakage achieving uniform intra-tumor distribution (UITD)-guided photothermal therapy

Photothermal therapy (PTT) has received increasing attention for treating tumors. However, a long-standing challenge in PTT is non-uniform distribution of photothermal agents (PAs) in tumor tissues, resulting in limited therapeutic efficiency. Herein, inspired by dandelions blowing away by the wind, we have designed a DNA-assembled visible GRS-DNA-CuS nanodandelion, which can achieve uniform intra-tumor distribution (UITD) of PAs, thus enhancing the photothermal therapeutic efficiency. GRS-DNA-CuS is featured by the formation of hydrogen bond between the core of single-strand DNA-modified Raman nanoprobes (GRS) and the shell of complementary single-strand DNA-modified CuS PAs. Under Raman imaging-guided 1st NIR irradiation, hydrogen bond in GRS-DNA-CuS is explosively broken, resulting in large-sized GRS-DNA-CuS (∼135 nm) be completely dissociated into GRS and ultra-small CuS PAs (∼12 nm) within 1 min. Such an explosive dissociation instantly enhances the local concentration of ultra-small CuS PAs and slightly rises intra-tumor temperature, thus increasing the diffusion coefficient of PAs and promoting their UITD. This UITD of CuS PAs enhances the photothermal anti-tumor effects. Three out of five tumors are completely eliminated under photoacoustic imaging-guided 2nd NIR irradiation. Overall, this study provides one UITD-guided PTT strategy for highly effective tumor treatment by exerting explosive breakage property of hydrogen bond, broadening the application scope of DNA-assembly technique in oncology field.

Preclinical and exploratory human studies of novel 68Ga-labeled D-peptide antagonist for PET imaging of TIGIT expression in cancers.

PurposeWhile TIGIT has been propelled as a next-generation target in cancer immunotherapy, anti-TIGIT therapy seems to be promising for a fraction of patients in clinical trials. Therefore, patient stratification is critical for this therapy, which could benefit from a whole-body, non-invasive, and quantitative evaluation of TIGIT expression in cancers. In this study, a 68Ga-labeled D-peptide antagonist, 68Ga-GP12, was developed and validated for PET imaging of TIGIT expression in vitro, in vivo, and in an exploratory human study.MethodsThe D-enantiomer peptide antagonists were modified and radiolabeled with 68Ga. In vitro binding assays were performed in human peripheral blood mononuclear cells (PBMCs) to assess their affinity and specificity. The imaging capacity, biodistribution, pharmacokinetics, and radiation dosimetry were investigated. Flow cytometry, autoradiography, and immunohistochemical staining were used to confirm the expression of TIGIT. The safety and potential of 68Ga-GP12 for PET/CT imaging of TIGIT expression were evaluated in NSCLC patients.Results68Ga-labeled D-peptides were conveniently produced with high radiochemical yields, radiochemical purities and molar activities. In vitro binding assays demonstrated 68Ga-GP12 has high affinity and specificity for TIGIT with a KD of 37.28 nM. In vivo and ex vivo studies demonstrated the capacity of 68Ga-GP12 for PET imaging of TIGIT expression with high tumor uptake of 4.22 ± 0.68 %ID/g and the tumor-to-muscle ratio of 12.94 ± 2.64 at 60 min post-injection. In NSCLC patients, primary and metastatic lesions found in 68Ga-GP12 PET images were comparable to that in 18F-FDG PET images. Moreover, tracer uptake in primary and metastatic lesions and intra-tumoral distribution in the large tumor were inhomogenous, indicating the heterogeneity of TIGIT expression.Conclusion68Ga-GP12 is a promising radiotracer for PET imaging of TIGIT expression in cancers, indicating its potential as a potential companion diagnostic for anti-TIGIT therapies.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00259-021-05672-x.

Engineered Mesenchymal Stem Cells as a Biotherapy Platform for Targeted Photodynamic Immunotherapy of Breast Cancer.

Interleukin-12 (IL12) is a pleiotropic cytokine with promising prospects for cancer immunotherapy. Though IL12 gene-based therapy can overcome the fatal hurdle of severe systemic toxicity, targeted delivery and tumor-located expression of IL12 gene remain the challenging issues yet to be solved. Photo-immunotherapy emerging as a novel and precise therapeutic strategy, which elaborately combines immune-activating agents with light-triggered photosensitizers for potentiated anticancer efficacy. Herein, an engineered stem cell-based biotherapy platform (MB/IL12-MSCs) incorporating immune gene plasmid IL12 (pIL12) and photosensitizer methylene blue (MB) is developed to realize tumor-homing delivery of therapeutic agents and photo-immunotherapy efficacy enhancement. The biotherapy platform retained tumor-tropic migration and penetration functions, which improved the intratumoral distribution of therapeutic agents, thereby promoting photodynamic effects and reinforcing immune responses. Importantly, MB/IL12-MSCs restricted the expression and distribution of IL12 at tumor site, which minimized potential toxicity while eliciting sufficient anticancer immunity. In noteworthy, activation of immunity induced by MB/IL12-MSCs established long-term systemic immunologic memory to prevent tumor relapse. The MB/IL12-MSCs outperform their monotherapy counterparts in breast tumor models, and the growth of tumor significantly arrested as well as re-challenging abscopal tumor growth slowdown. Collectively, this work reveals that MSCs-based strategy may advance more efficient, durable, and safer cancer photo-immunotherapy.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level.

CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro- and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D µComputer tomography bioluminescence tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining.Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (± IL-2) or control BDCA-2 CAR T cell (± IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D µCT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM.Results: 3D µCT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect.Conclusion: Overall, we demonstrated that 3D µCT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.

Real-time in vivo imaging reveals specific nanoparticle target binding in a syngeneic glioma mouse model.

Nanomaterial-based drug delivery is a promising strategy for glioma treatment. However, the detailed dynamics of nanoparticles in solid glioma are still a mystery, including their intratumoral infiltration depth, penetration, retention time, and distribution. Revealing these processes in detail requires repeated intravital imaging of the corresponding brain tumor regions over time during glioma growth. Hereby, we established a syngeneic orthotopic cerebral glioma mouse model by combining the chronic cranial window and two-photon microscopy. Thus, we were able to investigate the dynamics of the nanoparticles during long-term glioma growth. Three hours after the intravenous (i.v.) injection of integrin αVβ3 binding conjugated silicon nanoparticles (SNPs-PEG-RGD-FITC), green nanoparticles had already infiltrated the brain glioma, and then more nanoparticles penetrated into the solid brain tumor and were retained for at least 8 days. However, the amount of control SNPs-PEG-FITC that infiltrated into the solid brain tumor was very low. Moreover, we found that SNPs-PEG-RGD-FITC were not only located in the tumor border but could also infiltrate into the core region of the solid tumor. In vitro assay also confirmed the high binding affinity between GL-261-Tdtomato cells and SNPs-PEG-RGD-FITC. Our results indicate that SNPs-PEG-RGD-FITC has high penetration and retention in a solid glioma and our model provides novel ideas for the investigation of nanoparticle dynamics in brain tumors.

ОПТИМIЗАЦIЯ ХIМIОТЕРАПIЇ ЗЛОЯКIСНИХ ПУХЛИН НАОСНОВI ДОСТАВКИ ЛIКIВ IЗ ПIДСИЛЕНОЮКОНВЕКЦIЄЮ

The article describes a numerical method for optimizing the chemotherapy of malignant tumors on the basis of drug delivery using increased convection. The problem of optimal control with point sources for reaching the desired intratumor distribution of drugs in macroscopic scale granting the properties of intersticial space and effects of convective diffusion is considered. The efficiency of proposed algorithm for optimal control is shown.

RAS-Driven Macropinocytosis of Albumin or Dextran Reveals Mutation-Specific Target Engagement of RAS p.G12C Inhibitor ARS-1620 by NIR-Fluorescence Imaging.

Macropinocytosis serves as a highly conserved endocytotic process that has recently been shown as a critical mechanism by which RAS-transformed cells transport extracellular protein into intracellular amino acid pathways to support their unique metabolic needs. We developed NIR fluorescently labeled molecular imaging probes to monitor macropinocytosis-mediated uptake of albumin in a K-RAS-dependent manner. Using western blot analysis, immunofluorescence, and flow cytometry, albumin retention was characterized in vitro across several RAS-activated lung and pancreatic cancer cell lines. AF790-albumin was synthesized and administered to mice bearing K-RAS mutant xenograft tumors of H460 (K-RAS p.Q61H) and H358 (K-RAS p.G12C) non-small cell lung cancers on each flank. Mice were treated daily with 2mg/kg of ARS-1620, a targeted RAS p.G12C inhibitor, for 2days and imaged following each treatment. Subsequently, the mice were then treated daily with 10mg/kg of amiloride, a general inhibitor of macropinocytosis, for 2days and imaged. Intratumoral distribution of AF790-albumin was assessed in vivo using near-infrared (NIR) fluorescence imaging. Albumin retention was observed as a function of K-RAS activity and macropinocytosis across several lung and pancreatic cancer cell lines. We documented that ARS-1620-induced inhibition of K-RAS activity or amiloride-mediated inhibition of macropinocytosis significantly reduced albumin uptake. Tumor retention in vivo of AF790-albumin was both RAS inhibition-dependent as well as abrogated by inhibition of macropinocytosis. These data provide a novel approach using NIR-labeled human serum albumin to identify and monitor RAS-driven tumors as well as evaluate the on-target efficacy in vivo of inhibitors, such as ARS-1620.

Elucidation for Intratumor Localization of a DDS-based Anticancer Drug and Enhancement of Its Therapeutic Effects via Improvement of the Tumor Microenvironment

In the development of drug delivery system (DDS)-based anticancer drugs, the techniques for the intratumor mapping and quantification of active pharmaceutical ingredients (API) in pharmaceuticals must be pivotal for predicting pharmacological effects and adverse events. X-ray fluorescence spectrometry (XRF) is a potent analytical tool for mapping/quantifying platinum pharmaceutics such as oxaliplatin (l-OHP) and its liposomal formulation. In recent studies, we employed XRF to visualize the intratumor micro-distribution of l-OHP in a tumor-bearing model mouse intravenously injected with either free l-OHP or l-OHP liposomes. The intratumor distribution of l-OHP within tumor sections could be determined by XRF to detect platinum atoms. After treatment with the liposomal formulation, the l-OHP was localized near the tumor vessels and, via repeated injections, increasingly accumulated in tumors by a much greater degree than treatment with free l-OHP. The repeated injections of l-OHP liposomes improved the vascular permeability via inducing the apoptosis of tumor cells near the tumor vessels, which should improve the tumor microenvironment and enhance the intratumor accumulation of repeated doses of l-OHP liposomes. The proposed process was also used to visualize the intratumor distribution of l-OHP in rectal cancer specimens resected from a patient who had received l-OHP-based preoperative chemotherapy. We further revealed that neutralization of an acidic tumor microenvironment via oral administration with NaHCO3 could improve the therapeutic efficacy of weakly basic anticancer agent-encapsulating liposomes. Collectively, mapping/quantifying the intratumor API in DDS drugs and/or improving the tumor microenvironment would be an effective means to accelerate the clinical development of DDS-based anticancer drugs.

Spatial Profiles of Intratumoral PD-1+ Helper T Cells Predict Prognosis in Head and Neck Squamous Cell Carcinoma.

BackgroundFunctional interactions between immune cells and neoplastic cells in the tumor immune microenvironment have been actively pursued for both biomarker discovery for patient stratification, as well as therapeutic anti-cancer targets to improve clinical outcomes. Although accumulating evidence indicates that intratumoral infiltration of immune cells has prognostic significance, limited information is available on the spatial infiltration patterns of immune cells within intratumoral regions. This study aimed to understand the intratumoral heterogeneity and spatial distribution of immune cell infiltrates associated with cell phenotypes and prognosis in head and neck squamous cell carcinoma (HNSCC).MethodsA total of 88 specimens of oropharyngeal squamous cell carcinoma, categorized into discovery (n = 38) and validation cohorts (n = 51), were analyzed for immune contexture by multiplexed immunohistochemistry (IHC) and image cytometry-based quantification. Tissue segmentation was performed according to a mathematical morphological approach using neoplastic cell IHC images to dissect intratumoral regions into tumor cell nests versus intratumoral stroma.ResultsTissue segmentation revealed heterogeneity in intratumoral T cells, varying from tumor cell nest-polarized to intratumoral stroma-polarized distributions. Leukocyte composition analysis revealed higher ratios of TH1/TH2 in tumor cell nests with higher percentages of helper T cells, B cells, and CD66b+ granulocytes within intratumoral stroma. A discovery and validation approach revealed a high density of programmed death receptor-1 (PD-1)+ helper T cells in tumor cell nests as a negative prognostic factor for short overall survival. CD163+ tumor-associated macrophages (TAM) provided the strongest correlation with PD-1+ helper T cells, and cases with a high density of PD-1+ helper T cells and CD163+ TAM had a significantly shorter overall survival than other cases.ConclusionThis study reveals the significance of analyzing intratumoral cell nests and reports that an immune microenvironment with a high density of PD-1+ helper T cells in tumoral cell nests is a poor prognostic factor for HNSCC.