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Abstract Liver cancer is the second leading cause of cancer-related deaths worldwide, motivating major scientific efforts to understand and treat this cancer type. Over 30% of patients with liver cancer progress to metastasis, which reduces the survival rate. Extensive studies on primary tumors have been conducted to improve the prognosis. However, there is a lack of appropriate and accessible models for studying the progression and metastasis of liver cancer. Moreover, conventional metastasis models do not reproduce metastasis as it occurs in patients. To address this lack of an appropriate model for the monitoring of cancer progression and evaluation of anticancer drugs, we established a spontaneous metastatic xenograft model using NSG mice subcutaneously transplanted with SK-Hep-1 cells. Compared to the conventional experimental metastasis model (intravenous transplantation), in which only lung metastasis was observed, the established spontaneous metastatic xenograft model was superior, as it showed both a primary tumor and metastatic patterns similar to those observed in human patients. Additionally, this model was successfully used to assess the antimetastatic efficacy of sorafenib. In conclusion, our results demonstrate that the established spontaneous metastatic xenograft model better reflects liver cancer metastasis and can be utilized to assess the efficacy of anticancer drugs for liver cancer.

Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs which have demonstrated limited effectiveness in OC patients. We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). In the SKOV-3 Luc Hu-mouse model, we assessed the efficacy of PD-1 blockade with pembrolizumab. We observed the presence of human lymphocyte and myeloid cell subsets within the tumors, lymph nodes, blood, and spleens in these models. Notably, these tumors exhibited a high prevalence of tumor-infiltrating macrophages. Furthermore, we identified HDAC class I target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts in the tumors of Hu-mice treated with pembrolizumab. Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer useful information to explore potential mechanisms associated with unresponsive anti-PD-1 treatment in OC. [ABSTRACT FROM AUTHOR]

Guided Bone Regeneration is a common procedure, yet, as new grafting materials are being introduced into the market, a reliable evaluation method is required. Critical size defect in animal models provides an accurate simulation, followed by histological sections to evaluate the new bone formation. However, histology is destructive, two-dimensional and technique-sensitive. In this study we developed a novel volumetric Micro-CT analysis to quantify new bone formation characteristics. Eight adult female New Zealand white rabbits were subjected to calvarial critical-size defects. Four 8 mm in diameter circular defects were preformed in each animal, to allow random allocation of four treatment modalities. All calvarias were scanned using Micro-CT. Each defect was segmented into four equal parts: pristine bone, outer, middle, and inner. Amira software (v. 6.3, www.fei.com) was used to calculate the new bone volume in each region and compare it to that of the pristine bone. All grafting materials demonstrated that new bone formation decreased as it moved inward. Only the inner region differed across grafting materials (p = 0.001). The new Micro-CT analysis allowed us to divide each defect into 3D regions providing better understanding of the bone formation process. Amongst the various advantages of the Micro-CT, it enables us to quantify the graft materials and the newly formed bone independently, and to describe the defect morphology in 3D (bi- vs. uni-cortical defects). Providing an insight into the inner region of the defect can better predict the regenerative potential of the bone augmentation graft material. Therefore, the suggested Micro-CT analysis is beneficial for further developing of clinical approaches. [ABSTRACT FROM AUTHOR]

Patient-derived xenograft (PDX) tumor models are known to maintain the genomic and phenotypic profiles, including the histopathological structures, of the parental tumors. On the other hand, unique enrichment of single-nucleotide variants or copy number aberrations has been reported in several types of tumors. However, an understanding of endometrial carcinoma PDXs is limited. The purpose of the present study was to clarify the presence or absence of the molecular properties of endometrial carcinomas in PDXs passaged up to eight times. Established PDXs of endometrioid carcinomas maintained their histopathological characteristics, but those of carcinosarcomas predominantly consisted of sarcomatous components when compared to the parental tumors. Alterations in the proportion of cells with positive/negative immunohistochemical staining for estrogen receptor, PTEN, PAX8, and PAX2 were observed, whereas the proportions of cells with AE1/AE3, TP53, ARID1A, PMS2, and MSH6 staining were unchanged. Variants of cancer-associated genes were compared between PDXs and parental tumors. Mutations in POLE and a frameshift deletion in BRCA1 were observed in the parental tumor tissue in each of the six cases, and additional genomic alterations, which were not apparently related to histopathological and immunohistochemical alterations, were found in the PDXs of these cases. The genomic and phenotypic alterations observed between endometrial carcinoma PDXs and parental tumors were partly associated with endometrial cancer-specific characteristics related to cellular differentiation and gene mutations. [ABSTRACT FROM AUTHOR]

Pinus elliottii used as rootstock instead of homologous rootstock, have been proved to accelerate early growth of the scion (Pinus massoniana), for cultivation of large diameter wood. However, the basal diameter of scions in heterologous grafts was significantly smaller than self-graft 10 years later, according to field investigation, which was opposed to cultivation objectives. Although advantage of heterologous grafts has been reported, less is known about the long term effect of heterologous rootstock on scions of P. massoniana. The aim of present study was to investigate the mechanism of the above difference. Toward this aim, the growth traits and physiological characteristics of scions in the two graft groups were studied, and the underlying mechanism was preliminarily explored through transcriptome sequencing technology. Results showed that scions of heterologous grafts had less TSCA compared to self-grafts, while no significant difference of plant height, number of branches and canopy volume between two graft groups. Besides, scion leaves of heterologous grafts displayed higher antioxidant enzyme activity and lower chlorophyll content. And interactions between rootstocks and scions had also changed the mineral element composition of scion leaves. Compared with homologous grafts, scion leaves of heterologous grafts accumulated more K+, Mg2+ and Zn2+, but less Ca2+,which have been proved to be conducive to the growth of stem diameter of P. massoniana. Moreover, a comparative transcriptome analysis of two graft groups showed that DEGs between them were mainly caused by the specificity of rootstock. GO and KEGG analysis found that heterologous rootstock had different gene expression preferences, and the gene expression level between rootstocks and scions were significantly different, such as auxin auxin-related genes and stress responsive genes. That may imply that auxin pathway played an important role not only in grafting healing process, but also in maintaining the growth between scion and stock. Summary of all above results, we concluded that the long term effect of heterologous rootstock on scions may be unsatisfactory with the later rapidly growth of scion, probably due to delayed graft incompatibility between scion and stock of heterologous grafts. This study may remind us that the long-term growth of the scion deserves attention as well as the healing process, which could also provide a basis for delayed graft incompatibility. [ABSTRACT FROM AUTHOR]

Tumour cells exhibit altered metabolic pathways that lead to radiation resistance and disease progression. Raman spectroscopy (RS) is a label-free optical modality that can monitor post-irradiation biomolecular signatures in tumour cells and tissues. Convolutional Neural Networks (CNN) perform automated feature extraction directly from data, with classification accuracy exceeding that of traditional machine learning, in cases where data is abundant and feature extraction is challenging. We are interested in developing a CNN-based predictive model to characterize clinical tumour response to radiation therapy based on their degree of radiosensitivity or radioresistance. In this work, a CNN architecture is built for identifying post-irradiation spectral changes in Raman spectra of tumour tissue. The model was trained to classify irradiated versus non-irradiated tissue using Raman spectra of breast tumour xenografts. The CNN effectively classified the tissue spectra, with accuracies exceeding 92.1% for data collected 3 days post-irradiation, and 85.0% at day 1 post-irradiation. Furthermore, the CNN was evaluated using a leave-one-out- (mouse, section or Raman map) validation approach to investigate its generalization to new test subjects. The CNN retained good predictive accuracy (average accuracies 83.7%, 91.4%, and 92.7%, respectively) when little to no information for a specific subject was given during training. Finally, the classification performance of the CNN was compared to that of a previously developed model based on group and basis restricted non-negative matrix factorization and random forest (GBR-NMF-RF) classification. We found that CNN yielded higher classification accuracy, sensitivity, and specificity in mice assessed 3 days post-irradiation, as compared with the GBR-NMF-RF approach. Overall, the CNN can detect biochemical spectral changes in tumour tissue at an early time point following irradiation, without the need for previous manual feature extraction. This study lays the foundation for developing a predictive framework for patient radiation response monitoring. [ABSTRACT FROM AUTHOR]

This analysis determined in our case that the I first-order i features of I Skewness i and I Kurtosis i were robust to variations of grey levels and reconstruction analysis parameters, as well as wavelength chosen during image acquisition, while remaining sensitive enough to discriminate two breast PDX models of two different breast cancer subtypes. These authors contributed equally: Lorena Escudero Sanchez and Emma Brown. This analysis determines that the I first-order i features of I Skewness i and I Kurtosis i are robust to variations of grey levels and reconstruction analysis parameters, as well as wavelength chosen during image acquisition, while remaining sensitive to discriminate two breast PDX models of two different breast cancer subtypes. [Extracted from the article]

Prostate cancer (PCa) is a complex disease progressing from in situ to invasive or metastatic tumors while also being capable of modulating its androgen dependence. Understanding how novel therapies are working across the different stages of the disease is critical for their proper positioning in the spectrum of PCa treatments. The targeting of proprotein convertase PACE4 (Paired basic Amino Acid-Cleaving Enzyme 4) has been proposed as a novel approach to treat PCa. Animal studies performed on LNCaP xenografts, an androgen-dependent model, already yielded positive results. In this study, we tested PACE4 inhibition on JHU-LNCaP-SM, a newly described androgen-independent model, in cell-based and xenograft assays. Like LNCaP, JHU-LNCaP-SM cells express PACE4 and its oncogenic isoform PACE4-altCT. Using isoform-specific siRNAs, downregulation of PACE4-altCT resulted in JHU-LNCaP-SM growth inhibition. Furthermore, JHU-LNCaP-SM responded to the PACE4 pharmacological inhibitor known as C23 in cell-based assays as well as in athymic nude mice xenografts. These data support the efficacy of PACE4 inhibitors against androgen independent PCa thereby demonstrating that PACE4 is a key target in PCa. The JHU-LNCaP-SM cell line represents a model featuring important aspects of androgen-independent PCa, but it also represents a very convenient model as opposed to LNCaP cells for in vivo studies, as it allows rapid screening due to its high implantation rate and growth characteristics as xenografts. [ABSTRACT FROM AUTHOR]

Photoacoustic imaging is an increasingly popular method of exploring the tumour microenvironment, which can provide insight into tumour oxygenation status and potentially treatment response assessment. Currently, the measurements most commonly performed on such images are the mean and median of the pixel values of the tumour volumes of interest. We investigated expanding the set of measurements that can be extracted from these images by adding radiomic features. In particular, we found that Skewness was sensitive to differences between basal and luminal patient derived xenograft cancer models with an η 2 of 0.86, and that it was robust to variations in confounding factors such as reconstruction type and wavelength. We also built discriminant models with radiomic features that were correlated with the underlying tumour model and were independent from each other. We then ranked features by their importance in the model. Skewness was again found to be an important feature, as were 10th Percentile, Root Mean Squared, and several other texture-based features. In summary, this paper proposes a methodology to select radiomic features extracted from photoacoustic images that are robust to changes in acquisition and reconstruction parameters, and discusses features found to have discriminating power between the underlying tumour models in a pre-clinical dataset. [ABSTRACT FROM AUTHOR]

Anticancer combination therapy has been developed to increase efficacy by enhancing synergy. Patient-derived xenografts (PDXs) have emerged as reliable preclinical models to develop effective treatments in translational cancer research. However, most PDX combination study designs focus on single dose levels, and dose–response surface models are not appropriate for testing synergism. We propose a comprehensive statistical framework to assess joint action of drug combinations from PDX tumor growth curve data. We provide various metrics and robust statistical inference procedures that locally (at a fixed time) and globally (across time) access combination effects under classical drug interaction models. Integrating genomic and pharmacological profiles in non-small-cell lung cancer (NSCLC), we have shown the utilities of combPDX in discovering effective therapeutic combinations and relevant biological mechanisms. We provide an interactive web server, combPDX (https://licaih.shinyapps.io/CombPDX/), to analyze PDX tumor growth curve data and perform power analyses. [ABSTRACT FROM AUTHOR]

Given our interest in the utility of liposomes for molecular imaging and theranostics, we investigated how coating the outer layer of the liposome affects internalization by breast cancer cell lines in vitro and in breast tumor tissues in vivo. Indeed, we discovered that a remarkably high liposomal uptake can be achieved by DBCO (dibenzocyclooctyne) soft coating. Our data demonstrates that decorating the terminal lipid with a DBCO moiety at a specific density induces increased tumor uptake in vivo (tumor uptake ~ 50%) compared to conventional undecorated liposome (tumor uptake ~ 20%). In this study, we report improved visualization of breast cancer cells in vivo using a 4T1 orthotopic breast cancer model and primary breast tumor xenograft models MDA-MB-231 and MDA-MB-436. L-PEG2000-DBCO coated liposomes demonstrate increased accumulation in breast cancer cells independent of tumor size, type, position, receptor expression, as well as the condition of the host mice. We expect these findings to have a major positive impact on the practical utility of liposomes in image-guided applications and precision medicine theranostics. [ABSTRACT FROM AUTHOR]

RECK encodes a membrane-anchored protease-regulator which is often downregulated in a wide variety of cancers, and reduced RECK expression often correlates with poorer prognoses. In mouse models, forced expression of RECK in tumor xenografts results in suppression of tumor angiogenesis, invasion, and metastasis. RECK mutations, however, are rare in cancer genomes, suggesting that agents that re-activate dormant RECK may be of clinical value. We found a potent RECK-inducer, DSK638, that inhibits spontaneous lung metastasis in our mouse xenograft model. Induction of RECK expression involves SP1 sites in its promoter and may be mediated by KLF2. DSK638 also upregulates MXI1, an endogenous MYC-antagonist, and inhibition of metastasis by DSK638 is dependent on both RECK and MXI1. This study demonstrates the utility of our approach (using a simple reporter assay followed by multiple phenotypic assays) and DSK638 itself (as a reference compound) in finding potential metastasis-suppressing drugs. [ABSTRACT FROM AUTHOR]

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Among the key challenges in developing effective therapeutics is the poor translation of preclinical models used in the drug discovery pipeline. This leaves drug attrition rates and costs at an unacceptably high level. Previous work has highlighted the discrepancies in therapeutic response between current in vitro and in vivo models. To address this, we conducted a comparison study to differentiate the carboplatin chemotherapy response across four different model systems including 2D monolayers, 3D spheroids, 3D ex vivo tumors and mouse xenograft models. We used six previously characterized EOC cell lines of varying chemosensitivity and performed viability assays for each model. In vivo results from the mouse model correlated with 2D response in 3/6 cell lines while they correlated with 3D spheroids and the ex vivo model in 4/6 and 5/5 cell lines, respectively. Our results emphasize the variability in therapeutic response across models and demonstrate that the carboplatin response in EOC cell lines cultured in a 3D ex vivo model correlates best with the in vivo response. These results highlight a more feasible, reliable, and cost-effective preclinical model with the highest translational potential for drug screening and prediction studies in EOC. [ABSTRACT FROM AUTHOR]

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody–drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen. [ABSTRACT FROM AUTHOR]

[68Ga]PSMA-11 is a prostate-specific membrane antigen (PSMA)-targeting radiopharmaceutical for diagnostic PET imaging. Its application can be extended to targeted radionuclide therapy (TRT). In this study, we characterize the biodistribution and pharmacokinetics of [68Ga]PSMA-11 in PSMA-positive and negative (22Rv1 and PC3, respectively) tumor-bearing mice and subsequently estimated its internal radiation dosimetry via voxel-level dosimetry using a dedicated Monte Carlo simulation to evaluate the absorbed dose in the tumor directly. Consequently, this approach overcomes the drawbacks of the conventional organ-level (or phantom-based) method. The kidneys and urinary bladder both showed substantial accumulation of [68Ga]PSMA-11 without exhibiting a washout phase during the study. For the tumor, a peak concentration of 4.5 ± 0.7 %ID/g occurred 90 min after [68Ga]PSMA-11 injection. The voxel- and organ-level methods both determined that the highest absorbed dose occurred in the kidneys (0.209 ± 0.005 Gy/MBq and 0.492 ± 0.059 Gy/MBq, respectively). Using voxel-level dosimetry, the absorbed dose in the tumor was estimated as 0.024 ± 0.003 Gy/MBq. The biodistribution and pharmacokinetics of [68Ga]PSMA-11 in various organs of subcutaneous prostate cancer xenograft model mice were consistent with reported data for prostate cancer patients. Therefore, our data supports the use of voxel-level dosimetry in TRT to deliver personalized dosimetry considering patient-specific heterogeneous tissue compositions and activity distributions. [ABSTRACT FROM AUTHOR]

Specifying the exact localization of insulinoma remains challenging due to the lack of insulinoma-specific imaging methods. Recently, glucagon-like peptide-1 receptor (GLP-1R)-targeted imaging, especially positron emission tomography (PET), has emerged. Although various radiolabeled GLP-1R agonist exendin-4-based probes with chemical modifications for PET imaging have been investigated, an optimal candidate probe and its scanning protocol remain a necessity. Thus, we investigated the utility of a novel exendin-4-based probe conjugated with polyethylene glycol (PEG) for [18F]FB(ePEG12)12-exendin-4 PET imaging for insulinoma detection. We utilized [18F]FB(ePEG12)12-exendin-4 PET/CT to visualize mouse tumor models, which were generated using rat insulinoma cell xenografts. The probe demonstrated high uptake value on the tumor as 37.1 ± 0.4%ID/g, with rapid kidney clearance. Additionally, we used Pdx1-Cre;Trp53R172H;Rbf/f mice, which developed endogenous insulinoma and glucagonoma, since they enabled differential imaging evaluation of our probe in functional pancreatic neuroendocrine neoplasms. In this model, our [18F]FB(ePEG12)12-exendin-4 PET/CT yielded favorable sensitivity and specificity for insulinoma detection. Sensitivity: 30-min post-injection 66.7%, 60-min post-injection 83.3%, combined 100% and specificity: 30-min post-injection 100%, 60-min post-injection 100%, combined 100%, which was corroborated by the results of in vitro time-based analysis of internalized probe accumulation. Accordingly, [18F]FB(ePEG12)12-exendin-4 is a promising PET imaging probe for visualizing insulinoma. [ABSTRACT FROM AUTHOR]

Medulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma. [ABSTRACT FROM AUTHOR]

Lanatoside C has a promising anti-tumor activity and is a potential candidate for radiosensitizers. In this study, we have investigated the therapeutic efficacy of the combination of 131I-trastuzumab and lanatoside C for inhibition of human epidermal growth factor receptor 2 (HER2) positive tumor progression in NCI-N87 xenograft model. The combination treatment (131I-trastuzumab and lanatoside C) showed highest cytotoxicity when compared to non-treated control or trastuzumab alone or 131I alone or 131I-trastuzumab alone in vitro. Biodistribution studies using 131I-trastuzumab or combination of 131I-trastuzumab and lanatoside C showed tumor uptake in BALB/c nude mice bearing HER2 positive NCI-N87 tumor xenograft model. The higher tumor uptake was observed in 131I-trastuzumab (19.40 ± 0.04% ID/g) than in the combination of 131I-trastuzumab and lanatoside C (14.02 ± 0.02% ID/g) at 24 h post-injection. Most importantly, an antitumor effect was observed in mice that received the combination of 131I-trastuzumab and lanatoside C (p = 0.009) when compared to control. In addition, mice received lanatoside C alone (p = 0.085) or 131I-trastuzumab alone (p = 0.160) did not significantly inhibit tumor progression compared with control. Taken together, our data suggest that combination of 131I-trastuzumab and lanatoside C might be a potential synergistic treatment for radioimmunotherapy to control the HER2 positive tumor. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems. [ABSTRACT FROM AUTHOR]

Drastic sensitivity enhancement of dynamic nuclear polarization is becoming an increasingly critical methodology to monitor real-time metabolic and physiological information in chemistry, biochemistry, and biomedicine. However, the limited number of available hyperpolarized 13C probes, which can effectively interrogate crucial metabolic activities, remains one of the major bottlenecks in this growing field. Here, we demonstrate [1-13C] N-acetyl cysteine (NAC) as a novel probe for hyperpolarized 13C MRI to monitor glutathione redox chemistry, which plays a central part of metabolic chemistry and strongly influences various therapies. NAC forms a disulfide bond in the presence of reduced glutathione, which generates a spectroscopically detectable product that is separated from the main peak by a 1.5 ppm shift. In vivo hyperpolarized MRI in mice revealed that NAC was broadly distributed throughout the body including the brain. Its biochemical transformation in two human pancreatic tumor cells in vitro and as xenografts differed depending on the individual cellular biochemical profile and microenvironment in vivo. Hyperpolarized NAC can be a promising non-invasive biomarker to monitor in vivo redox status and can be potentially translatable to clinical diagnosis. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors bortezomib and carfilzomib are approved for the treatment of multiple myeloma and mantle cell lymphoma and have demonstrated clinical efficacy for the treatment of acute lymphoblastic leukemia (ALL). The t(4;11)(q21;q23) chromosomal translocation that leads to the expression of MLL–AF4 fusion protein and confers a poor prognosis, is the major cause of infant ALL. This translocation sensitizes tumor cells to proteasome inhibitors, but toxicities of bortezomib and carfilzomib may limit their use in pediatric patients. Many of these toxicities are caused by on-target inhibition of proteasomes in non-lymphoid tissues (e.g., heart muscle, gut, testicles). We found that MLL–AF4 cells express high levels of lymphoid tissue-specific immunoproteasomes and are sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914, even in the presence of stromal cells. Inhibition of multiple active sites of the immunoproteasomes was required to achieve cytotoxicity against ALL. ONX-0914, an inhibitor of LMP7 (ß5i) and LMP2 (ß1i) sites of the immunoproteasome, and LU-102, inhibitor of proteasome ß2 sites, exhibited synergistic cytotoxicity. Treatment with ONX-0914 significantly delayed the growth of orthotopic ALL xenograft tumors in mice. T-cell ALL lines were also sensitive to pharmacologically relevant concentrations of ONX-0914. This study provides a strong rationale for testing clinical stage immunoproteasome inhibitors KZ-616 and M3258 in ALL. [ABSTRACT FROM AUTHOR]

Patient-derived xenograft (PDX) and their xenograft-derived organoid (XDO) models that recapitulate the genotypic and phenotypic landscape of patient cancers could help to advance research and lead to improved clinical management. PDX models were established from 276 pancreato-duodenal and biliary cancer resections. Initial, passage 0 (P0) engraftment rates were 59% (118/199) for pancreatic, 86% (25/29) for duodenal, and 35% (17/48) for biliary ductal tumors. Pancreatic ductal adenocarcinoma (PDAC), had a P0 engraftment rate of 62% (105/169). KRAS mutant and wild-type PDAC models were molecularly profiled, and XDO models were generated to perform initial drug response evaluations. Subsets of PDAC PDX models showed global copy number variants and gene expression profiles that were retained with serial passaging, and they showed a spectrum of somatic mutations represented in patient tumors. PDAC XDO models were established, with a success rate of 71% (10/14). Pathway activation of KRAS-MAPK in PDXs was independent of KRAS mutational status. Four wild-type KRAS models were characterized by one with EGFR (L747-P753 del), two with BRAF alterations (N486_P490del or V600E), and one with triple negative KRAS/EGFR/BRAF. Model OCIP256, characterized by BRAF (N486-P490 del), had activated phospho-ERK. A combination treatment of a pan-RAF inhibitor (LY3009120) and a MEK inhibitor (trametinib) effectively suppressed phospho-ERK and inhibited growth of OCIP256 XDO and PDX models. PDAC/duodenal adenocarcinoma have high success rates forming PDX/organoid and retaining their phenotypic and genotypic features. These models may be effective tools to evaluate novel drug combination therapies. [ABSTRACT FROM AUTHOR]

A high rate of glycolysis, one of the most common features of cancer, is used in positron emission tomography (PET) imaging to visualize tumor tissues using 18F-fluorodeoxyglucose (18F-FDG). Heterogeneous intratumoral distribution of 18F-FDG in tissues has been established in some types of cancer, and the maximum standardized uptake value (SUVmax) has been correlated with poor prognosis. However, the phenotype of cells that show high 18F-FDG accumulation in tumors remains unknown. Here, we combined quantitative micro-autoradiography with fluorescence immunohistochemistry to simultaneously visualize 18F-FDG distribution, the expression of multiple proteins, and hypoxic regions in the cancer microenvironment of a human A431 xenograft tumor in C.B-17/Icr-scid/scid mice. We found that the highest 18F-FDG accumulation was in cancer-derived cells undergoing epithelial-mesenchymal transition (EMT) in hypoxic regions, implicating these regions as a major contributor to increased glucose metabolism, as measured by 18F-FDG-PET. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC) is known for being very aggressive, heterogeneous and highly metastatic. The standard of care treatment is still chemotherapy, with adjacent toxicity and low efficacy, highlighting the need for alternative and more effective therapeutic strategies. Edelfosine, an alkyl-lysophospholipid, has proved to be a promising therapy for several cancer types, upon delivery in lipid nanoparticles. Therefore, the objective of this work was to explore the potential of edelfosine for the treatment of TNBC. Edelfosine nanoemulsions (ET-NEs) composed by edelfosine, Miglyol 812 and phosphatidylcholine as excipients, due to their good safety profile, presented an average size of about 120 nm and a neutral zeta potential, and were stable in biorelevant media. The ability of ET-NEs to interrupt tumor growth in TNBC was demonstrated both in vitro, using a highly aggressive and invasive TNBC cell line, and in vivo, using zebrafish embryos. Importantly, ET-NEs were able to penetrate through the skin barrier of MDA-MB 231 xenografted zebrafish embryos, into the yolk sac, leading to an effective decrease of highly aggressive and invasive tumoral cells' proliferation. Altogether the results demonstrate the potential of ET-NEs for the development of new therapeutic approaches for TNBC. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is a high-risk subtype of breast cancer with high capacity for metastasis and lacking of therapeutic targets. Our previous studies indicated that cystathionine γ-lyase (CSE) may be a new target related to the recurrence or metastasis of TNBC. Downregulation of CSE could inhibit the growth and metastasis of TNBC. The purpose of this study was to investigate the activity of the novel CSE inhibitor I194496 against TNBC in vivo and in vitro. The anticancer activity of I194496 in vitro were detected by MTS, EdU, and transwell assays. Methylene blue assay was used to determine the H2S level. Western blot was performed to analyze the expression of related pathway proteins. Xenograft tumors in nude mice were used to analyze the anticancer activity of I194496 in vivo. I194496 exerted potent inhibitory effects than l-propargylglycine (PAG, an existing CSE inhibitor) on human TNBC cells and possessed lower toxicity in normal breast epithelial Hs578Bst cells. I194496 reduced the activity and expression of CSE protein and the release of H2S in human TNBC cells. Meanwhile, the protein levels of PI3K, Akt, phospho (p)-Akt, Ras, Raf, p-ERK, p-Anxa2, STAT3, p-STAT3, VEGF, FAK, and Paxillin were decreased in human TNBC cells administrated with I194496. Furthermore, I194496 showed more stronger inhibitory effects on human TNBC xenograft tumors in nude mice. I194496 could inhibit the growth of human TNBC cells via the dual targeting PI3K/Akt and Ras/Raf/ERK pathway and suppress the metastasis of human TNBC cells via down-regulating Anxa2/STAT3 and VEGF/FAK/Paxillin signaling pathways. CSE inhibitor I194496 might become a novel and potential agent in the treatment of TNBC. [ABSTRACT FROM AUTHOR]

Repeated measures studies are frequently performed in patient-derived xenograft (PDX) models to evaluate drug activity or compare effectiveness of cancer treatment regimens. Linear mixed effects regression models were used to perform statistical modeling of tumor growth data. Biologically plausible structures for the covariation between repeated tumor burden measurements are explained. Graphical, tabular, and information criteria tools useful for choosing the mean model functional form and covariation structure are demonstrated in a Case Study of five PDX models comparing cancer treatments. Power calculations were performed via simulation. Linear mixed effects regression models applied to the natural log scale were shown to describe the observed data well. A straight growth function fit well for two PDX models. Three PDX models required quadratic or cubic polynomial (time squared or cubed) terms to describe delayed tumor regression or initial tumor growth followed by regression. Spatial(power), spatial(power) + RE, and RE covariance structures were found to be reasonable. Statistical power is shown as a function of sample size for different levels of variation. Linear mixed effects regression models provide a unified and flexible framework for analysis of PDX repeated measures data, use all available data, and allow estimation of tumor doubling time. [ABSTRACT FROM AUTHOR]

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is highly responsive to neoadjuvant polychemotherapy regimens including anthracyclines, taxanes, and, more recently, carboplatin. However, there is inadequate information on the individual contribution of each of these agents to the global activity of the combinations, and the use of combinations of up to four of these drugs is associated with relevant toxicity. Identifying single-drug activity in the clinical neoadjuvant setting is challenging. We developed patient-derived xenografts (PDXs) from several chemotherapy-naïve TNBC samples to assess the antitumor activity of single drugs and combinations of drugs. PDXs were established from chemotherapy-naïve TNBC samples. Nine TNBC PDX models (all of which corresponded to a basal-like phenotype according to the PAM50 classifier) were treated with carboplatin, docetaxel, and doxorubicin and the combination of docetaxel and carboplatin. Only one of nine PDX models showed sensitivity to doxorubicin, while eight of nine PDX models showed sensitivity to docetaxel and carboplatin as single agents. The 3 PDX models derived from patients with gBRCA-1 or gPALB2 mutations were very sensitive to carboplatin single agent. All 6 PDX models from patients without hereditary germ-line mutations showed increased sensitivity to the combination of docetaxel and carboplatin. In the present study, docetaxel and carboplatin single agents were active drugs against basal-like TNBC, while doxorubicin monotherapy showed low activity. The combination of docetaxel and carboplatin was more effective than the drugs used as single agents, except in the PDX from patients with gBRCA1/PALB2 mutations. [ABSTRACT FROM AUTHOR]

Personalized medicine holds tremendous promise for improving safety and efficacy of drug therapies by optimizing treatment regimens. Rapidly developed patient-derived xenografts (pdx) could be a helpful tool for analyzing the effect of drugs against an individual's tumor by growing the tumor in an immunodeficient animal. Severe combined immunodeficiency (SCID) mice enable efficient in vivo expansion of vital tumor cells and generation of personalized xenografts. However, they are not amenable to large-scale rapid screening, which is critical in identifying new compounds from large compound libraries. The development of a zebrafish model suitable for pdx could facilitate large-scale screening of drugs targeted against specific malignancies. Here, we describe a novel strategy for establishing a zebrafish model for drug testing in leukemia xenografts. We used chronic myelogenous leukemia and acute myeloid leukemia for xenotransplantation into SCID zebrafish to evaluate drug screening protocols. We showed the in vivo efficacy of the ABL inhibitor imatinib, MEK inhibitor U0126, cytarabine, azacitidine and arsenic trioxide. We performed corresponding in vitro studies, demonstrating that combination of MEK- and FLT3-inhibitors exhibit an enhanced effect in vitro. We further evaluated the feasibility of zebrafish for transplantation of primary human hematopoietic cells that can survive at 15 day-post-fertilization. Our results provide critical insights to guide development of high-throughput platforms for evaluating leukemia. [ABSTRACT FROM AUTHOR]

FGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib. [ABSTRACT FROM AUTHOR]

The protein alpha-synuclein (α-syn) is unusual because, depending on its conformation and the type of cell in which it is expressed, it is pro-death or pro-survival, triggering neurodegeneration in Parkinson's disease and enhancing cell survival of some melanomas. To probe the function of α-syn in melanoma, we used CRISPR/Cas9 to knockout SNCA, the gene that codes for α-syn, in SK-Mel-28 melanoma cells. The SNCA-knockout clones in culture exhibited a decrease in the transferrin receptor 1 (TfR1), an increase in ferritin, an increase of reactive oxygen species and proliferated slower than control cells. These SNCA-knockout clones grafted into SCID mice grew significantly slower than the SK-Mel-28 control cells that expressed α-syn. In the excised SNCA-knockout xenografts, TfR1 decreased 3.3-fold, ferritin increased 6.2-fold, the divalent metal ion transporter 1 (DMT1) increased threefold, and the iron exporter ferroportin (FPN1) decreased twofold relative to control xenografts. The excised SNCA-KO tumors exhibited significantly more ferric iron and TUNEL staining relative to the control melanoma xenografts. Collectively, depletion of α-syn in SK-Mel-28 cells dysregulates cellular iron metabolism, especially in xenografts, yielding melanoma cells that are deficient in TfR1 and FPN1, that accumulate ferric iron and ferritin, and that undergo apoptosis relative to control cells expressing α-syn. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of "pieces" of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ−/− (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation. [ABSTRACT FROM AUTHOR]

Cancer-associated fibroblasts (CAFs) are the key components of the densely proliferated stroma in pancreatic ductal adenocarcinoma (PDAC) and contribute to tumor progression and drug resistance. CAFs comprise heterogeneous subpopulations playing unique and vital roles. However, the commonly used mouse models have not been able to fully reproduce the histological and functional characteristics of clinical human CAF. Here, we generated a human cell-derived stroma-rich CDX (Sr-CDX) model, to reproduce the clinical tumor microenvironment. By co-transplanting human adipose-derived mesenchymal stem cells (AD-MSCs) and a human PDAC cell line (Capan-1) into mice, the Sr-CDX model recapitulated the characteristics of clinical pancreatic cancer, such as accelerated tumor growth, abundant stromal proliferation, chemoresistance, and dense stroma formed from the heterogeneous CAFs. Global RNA sequencing, single-cell based RNA sequencing, and histological analysis of CAFs in the Sr-CDX model revealed that the CAFs of the Sr-CDX mice were derived from the transplanted AD-MSCs and composed of heterogeneous subpopulations of CAF, including known and unknown subtypes. These lines of evidences suggest that our new tumor-bearing mouse model has the potential to address an open question in CAF research, that is the mechanism of CAF differentiation. [ABSTRACT FROM AUTHOR]

Bladder cancer is among the most prevalent cancers worldwide. Currently, few bladder cancer models have undergone thorough characterization to assess their fidelity to patient tumors, especially upon propagation in the laboratory. Here, we establish and molecularly characterize CoCaB 1, an aggressive cisplatin-resistant muscle-invasive bladder cancer patient-derived xenograft (PDX) and companion organoid system. CoCaB 1 was a subcutaneous PDX model reliably transplanted in vivo and demonstrated an acceleration in growth upon serial transplantation, which was reflected in organoid and 2D cell culture systems. Transcriptome analysis revealed progression towards an increasingly proliferative and stem-like expression profile. Gene expression differences between organoid and PDX models reflected expected differences in cellular composition, with organoids enriched in lipid biosynthesis and metabolism genes and deprived of extracellular components observed in PDXs. Both PDX and organoid models maintained the histological fidelity and mutational heterogeneity of their parental tumor. This study establishes the CoCaB 1 PDX and organoid system as companion representative tumor models for the development of novel bladder cancer therapies. [ABSTRACT FROM AUTHOR]

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) brain metastasis cell lines and in vivo models are not widely accessible. Herein we report on a direct-from patient-derived xenograft (PDX) model system of NSCLC brain metastases with genomic annotation useful for translational and mechanistic studies. Both heterotopic and orthotopic intracranial xenografts were established and RNA and DNA sequencing was performed on patient and matching tumors. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Transcriptome and mutation analysis revealed high correlation between matched patient and PDX samples with more than more than 95% of variants detected being retained in the matched PDXs. PDXs demonstrated response to radiation, response to selumetinib in tumors harboring KRAS G12C mutations and response to savolitinib in a tumor with MET exon 14 skipping mutation. Savolitinib also demonstrated in vivo radiation enhancement in our MET exon 14 mutated PDX. Early passage cell strains showed high consistency between patient and PDX tumors. Together, these data describe a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics. [ABSTRACT FROM AUTHOR]

We established patient-derived xenografts (PDX) from human primary breast cancers and studied whether stability or progressive events occurred during long-term in vivo passages (up to 4 years) in severely immunodeficient mice. While most PDX showed stable biomarker expression and growth phenotype, a HER2-positive PDX (PDX-BRB4) originated a subline (out of 6 studied in parallel) that progressively acquired a significantly increased tumor growth rate, resistance to cell senescence of in vitro cultures, increased stem cell marker expression and high lung metastatic ability, along with a strong decrease of BCL2 expression. RNAseq analysis of the progressed subline showed that BCL2 was connected to three main hub genes also down-regulated (CDKN2A, STAT5A and WT1). Gene expression of progressed subline suggested a partial epithelial-to-mesenchymal transition. PDX-BRB4 with its progressed subline is a preclinical model mirroring the clinical paradox of high level-BCL2 as a good prognostic factor in breast cancer. Sequential in vivo passages of PDX-BRB4 chronically treated with trastuzumab developed progressive loss of sensitivity to trastuzumab while HER2 expression and sensitivity to the pan-HER tyrosine kinase inhibitor neratinib were maintained. Long-term PDX studies, even though demanding, can originate new preclinical models, suitable to investigate the mechanisms of breast cancer progression and new therapeutic approaches. [ABSTRACT FROM AUTHOR]

To understand the potential effects of cancer cells on surrounding normal mammary epithelial cells, we performed direct co-culture of non-tumorigenic mammary epithelial MCF10A cells and various breast cancer cells. Firstly, we observed dynamic cell–cell interactions between the MCF10A cells and breast cancer cells including lamellipodia or nanotube-like contacts and transfer of extracellular vesicles. Co-cultured MCF10A cells exhibited features of epithelial-mesenchymal transition, and showed increased capacity of cell proliferation, migration, colony formation, and 3-dimensional sphere formation. Direct co-culture showed most distinct phenotype changes in MCF10A cells followed by conditioned media treatment and indirect co-culture. Transcriptome analysis and phosphor-protein array suggested that several cancer-related pathways are significantly dysregulated in MCF10A cells after the direct co-culture with breast cancer cells. S100A8 and S100A9 showed distinct up-regulation in the co-cultured MCF10A cells and their microenvironmental upregulation was also observed in the orthotropic xenograft of syngeneic mouse mammary tumors. When S100A8/A9 overexpression was induced in MCF10A cells, the cells showed phenotypic features of directly co-cultured MCF10A cells in terms of in vitro cell behaviors and signaling activities suggesting a S100A8/A9-mediated transition program in non-tumorigenic epithelial cells. This study suggests the possibility of dynamic cell–cell interactions between non-tumorigenic mammary epithelial cells and breast cancer cells that could lead to a substantial transition in molecular and functional characteristics of mammary epithelial cells. [ABSTRACT FROM AUTHOR]

Cisplatin, metformin, and quercetin are all reliable anticancer drugs. However, it is unclear how effective their different combination regimens are on the growth of nasopharyngeal carcinoma cell line Sune-1 and subcutaneous xenograft in nude mice. This study evaluated the effects of single-drug, two-drug, and three-drug simultaneous or sequential combined application of these drugs on the growth of Sune-1 cells and subcutaneous xenograft tumors in nude mice. The results showed that the different combination regimens of cisplatin, metformin and quercetin all had significant inhibitory effects on the proliferation of Sune-1 cells and the growth of subcutaneous xenografts in nude mice (P < 0.01), and the inhibition rate of the three drugs simultaneous combined application was significant Higher than the two-drug combination or single-drug application (P < 0.05), the contribution level of each drug in the three-drug combination application from high to low were cisplatin > metformin > quercetin. In summary, our results indicate that the simultaneous combination of cisplatin, metformin, and quercetin may synergistically inhibit the growth of Sune-1 cells and subcutaneous xenografts in nude mice through their different anticancer mechanisms, which may be clinically refractory and provide reference for chemotherapy of patients with recurrent nasopharyngeal carcinoma. [ABSTRACT FROM AUTHOR]

We xeno-transplanted human neural precursor cells derived from induced pluripotent stem cells into the cerebellum and brainstem of mice and rats during prenatal development or the first postnatal week. The transplants survived and started to differentiate up to 1 month after birth when they were rejected by both species. Extended survival and differentiation of the same cells were obtained only when they were transplanted in NOD-SCID mice. Transplants of human neural precursor cells mixed with the same cells after partial in vitro differentiation or with a cellular extract obtained from adult rat cerebellum increased survival of the xeno-graft beyond one month. These findings are consistent with the hypothesis that the slower pace of differentiation of human neural precursors compared to that of rodents restricts induction of immune-tolerance to human antigens expressed before completion of maturation of the immune system. With further maturation the transplanted neural precursors expressed more mature antigens before the graft were rejected. Supplementation of the immature cells suspensions with more mature antigens may help to induce immune-tolerance for those antigens expressed only later by the engrafted cells. [ABSTRACT FROM AUTHOR]

Tumor xenograft models can create a high capacity to study human tumors and discover efficient therapeutic approaches. Here, we aimed to develop the gamma-radiated immunosuppressed (GIS) mice as a new kind of tumor xenograft model for biomedical studies. First, 144 mice were divided into the control and treated groups exposed by a medical Cobalt-60 apparatus in 3, 4, and 5 Gy based on the system outputs. Then, 144 BALB/c mice were divided into four groups; healthy, xenograft, radiation, and radiation + xenograft groups. The animals in the xenograft and radiation + xenograft groups have subcutaneously received 3 × 106 MCF-7 cells 24 h post-radiation. On 3, 7, 14, and 21 days after cell injection, the animals were sacrificed. Then, the blood samples and the spleen and tumor tissues were removed for the cellular and molecular analyses. The whole-body gamma radiation had a high immunosuppressive effect on the BALB/c mice from 1 to 21 days post-radiation. The macroscopic and histopathological observations have proved that the created clusters' tumor structure resulted in the xenograft breast tumor. There was a significant increase in tumor size after cell injection until the end of the study. Except for Treg, the spleen level of CD4, CD8, CD19, and Ly6G was significantly decreased in Xen + Rad compared to the Xen alone group on 3 and 7 days. Unlike IL-4 and IL-10, the spleen level of TGF-β, INF-γ, IL-12, and IL-17 was considerably decreased in the Xen + Rad than the Xen alone group on 3 and 7 days. The spleen expressions of the VEGF, Ki67, and Bax/Bcl-2 ratio were dramatically increased in the Xen + Rad group compared to the Xen alone on 3, 7, 14, and 21 days. Our results could confirm a new tumor xenograft model via an efficient immune-suppressive potential of the whole-body gamma radiation in mice. [ABSTRACT FROM AUTHOR]

Histopathology is currently the most reliable tool in assessing the aggressiveness and prognosis of solid tumours. However, developing non-invasive modalities for tumour evaluation remains crucial due to the side effects and complications caused by biopsy procedures. In this study, saturation transfer MRI was used to investigate the microstructural and metabolic properties of tumour xenografts in mice derived from the prostate cancer cell lines 22Rv1 and DU145, which express different aggressiveness. The magnetization transfer (MT) and chemical exchange saturation transfer (CEST) effects, which are associated with the microstructural and metabolic properties in biological tissue, respectively, were analyzed quantitatively and compared amongst different tumour types and regions. Histopathological staining was performed as a reference. Higher cellular density and metabolism expressed in more aggressive tumours (22Rv1) were associated with larger MT and CEST effects. High collagen content in the necrotic regions might explain their higher MT effects compared to tumour regions. [ABSTRACT FROM AUTHOR]

Neuroblastoma resection represents a major challenge in pediatric surgery, because of the high risk of complications. Fluorescence-guided surgery (FGS) could lower this risk by facilitating discrimination of tumor from normal tissue and is gaining momentum in adult oncology. Here, we provide the first molecular-targeted fluorescent agent for FGS in pediatric oncology, by developing and preclinically evaluating a GD2-specific tracer consisting of the immunotherapeutic antibody dinutuximab-beta, recently approved for neuroblastoma treatment, conjugated to near-infrared (NIR) fluorescent dye IRDye800CW. We demonstrated specific binding of anti-GD2-IRDye800CW to human neuroblastoma cells in vitro and in vivo using xenograft mouse models. Furthermore, we defined an optimal dose of 1 nmol, an imaging time window of 4 days after administration and show that neoadjuvant treatment with anti-GD2 immunotherapy does not interfere with fluorescence imaging. Importantly, as we observed universal, yet heterogeneous expression of GD2 on neuroblastoma tissue of a wide range of patients, we implemented a xenograft model of patient-derived neuroblastoma organoids with differential GD2 expression and show that even low GD2 expressing tumors still provide an adequate real-time fluorescence signal. Hence, the imaging advancement presented in this study offers an opportunity for improving surgery and potentially survival of a broad group of children with neuroblastoma. [ABSTRACT FROM AUTHOR]

PP242, an inhibitor of mechanistic target of rapamycin (mTOR), displays potent anticancer effects against various cancer types. However, the underlying metabolic mechanism associated with the PP242 effects is not clearly understood. In this study, comprehensive metabolomics and lipidomics investigations were performed using ultra-high-performance chromatography-Orbitrap-mass spectrometry (UHPLC-Orbitrap-MS) in plasma and tumor tissue to reveal the metabolic mechanism of PP242 in an LS174T cell-induced colon cancer xenograft mouse model. After 3 weeks of PP242 treatment, a reduction in tumor size and weight was observed without any critical toxicities. According to results, metabolic changes due to the effects of PP242 were not significant in plasma. In contrast, metabolic changes in tumor tissues were very significant in the PP242-treated group compared to the xenograft control (XC) group, and revealed that energy and lipid metabolism were mainly altered by PP242 treatment like other cancer inhibitors. Additionally, in this study, it was discovered that not only TCA cycle but also fatty acid β-oxidation (β-FAO) for energy metabolism was inhibited and clear reduction in glycerophospholipid was observed. This study reveals new insights into the underlying anticancer mechanism of the dual mTOR inhibitor PP242, and could help further to facilitate the understanding of PP242 effects in the clinical application. [ABSTRACT FROM AUTHOR]

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs. [ABSTRACT FROM AUTHOR]

Understanding mechanisms mediating tumor metastasis is crucial for diagnostic and therapeutic targeting. Here, we take advantage of a transparent embryonic zebrafish xenograft model (eZXM) to visualize and track metastatic cells in real time using selective plane illumination microscopy (SPIM) for up to 30 h. Injected human leukemic and breast cancer cells exhibited cell-type specific patterns of intravascular distribution with leukemic cells moving faster than breast cancer cells. Tracking of tumor cells from high-resolution images revealed acute differences in intravascular speed and distance covered by cells. While the majority of injected breast cancer cells predominantly adhered to nearby vasculature, about 30% invaded the non-vascularized tissue, reminiscent of their metastatic phenotype. Survival of the injected tumor cells appeared to be partially inhibited and time-lapse imaging showed a possible role for host macrophages of the recipient embryos. Leukemic cell dissemination could be effectively blocked by pharmacological ROCK1 inhibition using Fasudil. These observations, and the ability to image several embryos simultaneously, support the use of eZXM and SPIM imaging as a functional screening platform to identify compounds that suppress cancer cell spread and invasion. [ABSTRACT FROM AUTHOR]

The unique microenvironment of the prostate plays a crucial role in the development and progression of prostate cancer (PCa). We examined the effects of cancer-associated fibroblasts (CAFs) on PCa progression using patient-derived fibroblast primary cultures in a representative orthotopic xenograft model. Primary cultures of CAFs, non-cancer-associated fibroblasts (NCAFs) and benign prostate hyperplasia-associated fibroblasts (BPHFs) were generated from patient-derived tissue specimens. These fibroblasts were coinjected together with cancer cells (LuCaP136 spheroids or LNCaP cells) in orthotopic PCa xenografts to investigate their effects on local and systemic tumor progression. Primary tumor growth as well as metastatic spread to lymph nodes and lungs were significantly stimulated by CAF coinjection in LuCaP136 xenografts. When NCAFs or BPHFs were coinjected, tumor progression was similar to injection of tumor cells alone. In LNCaP xenografts, all three fibroblast types significantly stimulated primary tumor progression compared to injection of LNCaP cells alone. CAF coinjection further increased the frequency of lymph node and lung metastases. This is the first study using an orthotopic spheroid culture xenograft model to demonstrate a stimulatory effect of patient-derived CAFs on PCa progression. The established experimental setup will provide a valuable tool to further unravel the interacting mechanisms between PCa cells and their microenvironment. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal cancer and is often refractory to current therapies. Development of efficient therapeutic strategies against ESCC presents a major challenge. Glycogen synthase kinase (GSK)3β has emerged as a multipotent therapeutic target in various diseases including cancer. Here we investigated the biology and pathological role of GSK3β in ESCC and explored the therapeutic effects of its inhibition. The expression of GSK3β and tyrosine (Y)216 phosphorylation-dependent activity was higher in human ESCC cell lines and primary tumors than untransformed esophageal squamous TYNEK-3 cells from an ESCC patient and tumor-adjacent normal esophageal mucosa. GSK3β-specific inhibitors and small interfering (si)RNA-mediated knockdown of GSK3β attenuated tumor cell survival and proliferation, while inducing apoptosis in ESCC cells and their xenograft tumors in mice. GSK3β inhibition spared TYNEK-3 cells and the vital organs of mice. The therapeutic effect of GSK3β inhibition in tumor cells was associated with G0/G1- and G2/M-phase cell cycle arrest, decreased expression of cyclin D1 and cyclin-dependent kinase (CDK)4 and increased expression of cyclin B1. These results suggest the tumor-promoting role of GSK3β is via cyclin D1/CDK4-mediated cell cycle progression. Consequently, our study provides a biological rationale for GSK3β as a potential therapeutic target in ESCC. [ABSTRACT FROM AUTHOR]