Research autopsy programmes in oncology: shared experience from 14 centres across the world.

While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

High-grade tumours promote growth of other less-malignant tumours in the same prostate.

Prostate cancer is a multifocal disease, but if and how individual prostate tumours influence each other is largely unknown. We therefore explored signs of direct or indirect tumour-tumour interactions in experimental models and patient samples. Low-metastatic AT1 and high-metastatic MatLyLu (MLL) Dunning rat prostate cancer cells were injected into separate lobes of the ventral prostate of immunocompetent rats. AT1 tumours growing in the same prostate as MLL tumours had increased tumour size and proliferation compared to AT1 tumours growing alone. In addition, the vasculature and macrophage density surrounding the AT1 tumours were increased by MLL tumour closeness. In patient prostatectomy samples, selected to contain an index tumour [tumour with the highest grade, International Society of Urological Pathology (ISUP) grade 1, 2, 3 or 4] and a low-grade satellite tumour (ISUP grade 1), cell proliferation in low-grade satellite tumours gradually increased with increasing histological grade of the index tumour. The density of blood vessels and CD68+ macrophages also increased around the low-grade satellite tumour if a high-grade index tumour was present. This suggests that high-grade tumours, by changing the prostate microenvironment, may increase the aggressiveness of low-grade lesions in the organ. Future studies are needed to explore the mechanisms behind tumour-tumour interactions and their clinical importance. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Canine transmissible venereal tumour established in immunodeficient mice reprograms the gene expression profiles associated with a favourable tumour microenvironment to enable cancer malignancy.

BACKGROUND: Canine transmissible venereal tumours (CTVTs) can cross the major histocompatibility complex barrier to spread among dogs. In addition to the transmissibility within canids, CTVTs are also known as a suitable model for investigating the tumour-host immunity interaction because dogs live with humans and experience the same environmental risk factors for tumourigenesis. Moreover, outbred dogs are more appropriate than inbred mice models for simulating the diversity of human cancer development. This study built a new model of CTVTs, known as MCTVTs, to further probe the shaping effects of immune stress on tumour development. For xenotransplantation, CTVTs were first injected and developed in immunodeficient mice (NOD.CB17-Prkdcscid/NcrCrl), defined as XCTVTs. The XCTVTs harvested from NOD/SCID mice were then inoculated and grown in beagles and named mouse xenotransplantation of CTVTs (MCTVTs). RESULTS: After the inoculation of CTVTs and MCTVTs into immune-competent beagle dogs separately, MCTVTs grew faster and metastasized more frequently than CTVTs did. Gene expression profiles in CTVTs and MCTVTs were analysed by cDNA microarray to reveal that MCTVTs expressed many tumour-promoting genes involved in chronic inflammation, chemotaxis, extracellular space modification, NF-kappa B pathways, and focal adhesion. Furthermore, several well-known tumour-associated biomarkers which could predict tumour progression were overexpressed in MCTVTs. CONCLUSIONS: This study demonstrated that defective host immunity can result in gene instability and enable transcriptome reprogramming within tumour cells. Fast tumour growth in beagle dogs and overexpression of tumour-associated biomarkers were found in a CTVT strain previously established in immunodeficient mice. In addition, dysregulated interaction of chronic inflammation, chemotaxis, and extracellular space modification were revealed to imply the possibly exacerbating mechanisms in the microenvironments of these tumours. In summary, this study offers a potential method to facilitate tumour progression and provide a niche for discovering tumour-associated biomarkers in cancer research.

Efficacy of punarnavine in restraining organ-specific tumour progression in 4T1-induced murine breast tumour model.

Most of the breast cancer deaths occur when cancer cells depart from their tumour of origin and spread systemically and colonise distant organs. The present study was to find out whether punarnavine, the quinolizidine alkaloid, with already proven antimetastatic effect on spontaneous B16F10 pulmonary metastasis has got any effect on a drastic organ-specific breast cancer spread. For the study, we selected a syngenic mouse 4T1 breast tumour model that mimics stage four of human breast cancer. The metastatic progression of 4T1 to lymph nodes, lungs, and liver was reduced by punarnavine (40 mg/kg body weight) administration in BALB/c mice. This was evident from the histopathology of these organs as well as from the reduction in the metastatic cell density of cultured 6-thioguanine-resistant 4T1 cells in the punarnavine-treated group compared to the control group. There was also a significant (p < 0.0001) inhibition of the primary breast tumour growth in the orthotopic site of induction with a simultaneous increase (p < 0.0001) in the life span of treated animals. The assessment of biochemical parameters such as hydroxyproline, hexosamine, uronic acid, sialic acid and γ-glutamyl transferase and the analysis of various cytokines VEGF, IL-1ß, TNF-α and GM-CSF showed a similar pattern of reduction in punarnavine (p < 0.0001) treated group compared to the control group. The gene expression study revealed the inhibitory effect of punarnavine on the major genes MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF involved in the metastatic process. These findings undeniably proved the potential of this quinolizidine alkaloid in combating breast tumour development and its progression in the studied murine model.

Engineered 3D tumour model for study of glioblastoma aggressiveness and drug evaluation on a detachably assembled microfluidic device.

3D models of tumours have emerged as an advanced technique in pharmacology and tumour cell biology, in particular for studying malignant tumours such as glioblastoma multiforme (GBM). Herein, we developed a 3D GBM model on a detachably assembled microfluidic device, which could be used to study GBM aggressiveness and for anti-GBM drug testing. Fundamental characteristics of the GBM microenvironment in terms of 3D tissue organisation, extracellular matrices and blood flow were reproduced in vitro by serial manipulations in the integrated microfluidic device, including GBM spheroid self-assembly, embedding in a collagen matrix, and continuous perfusion culture, respectively. We could realize multiple spheroids parallel manipulation, whilst, compartmentalized culture, in a highly flexible manner. This method facilitated investigations into the viability, proliferation, invasiveness and phenotype transition of GBM in a 3D microenvironment and under continuous stimulation by drugs. Anti-invasion effect of resveratrol, a naturally isolated polyphenol, was innovatively evaluated using this in vitro 3D GBM model. Temozolomide and the combination of resveratrol and temozolomide were also evaluated as control. This scalable model enables research into GBM in a more physiologically relevant microenvironment, which renders it promising for use in translational or personalised medicine to examine the impact of, or identify combinations of, therapeutic agents.

Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet.

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, details of the liver-specific molecular mechanisms responsible for the NAFLD-NASH-HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis-associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.

Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes.

BACKGROUND & AIMS: Concerns are raised about the representativeness of cell lines for tumours due to the culture environment and misidentification. Liver is a major metastatic destination of many cancers, which might further confuse the origin of hepatocellular carcinoma cell lines. Therefore, it is of crucial importance to understand how well they can represent hepatocellular carcinoma. METHODS: The HCC-specific gene pairs with highly stable relative expression orderings in more than 99% of hepatocellular carcinoma but with reversed relative expression orderings in at least 99% of one of the six types of cancer, colorectal carcinoma, breast carcinoma, non-small-cell lung cancer, gastric carcinoma, pancreatic carcinoma and ovarian carcinoma, were identified. RESULTS: With the simple majority rule, the HCC-specific relative expression orderings from comparisons with colorectal carcinoma and breast carcinoma could exactly discriminate primary hepatocellular carcinoma samples from both primary colorectal carcinoma and breast carcinoma samples. Especially, they correctly classified more than 90% of liver metastatic samples from colorectal carcinoma and breast carcinoma to their original tumours. Finally, using these HCC-specific relative expression orderings from comparisons with six cancer types, we identified eight of 24 hepatocellular carcinoma cell lines in the Cancer Cell Line Encyclopedia (Huh-7, Huh-1, HepG2, Hep3B, JHH-5, JHH-7, C3A and Alexander cells) that are highly representative of hepatocellular carcinoma. Evaluated with a REOs-based prognostic signature for hepatocellular carcinoma, all these eight cell lines showed the same metastatic properties of the high-risk metastatic hepatocellular carcinoma tissues. CONCLUSIONS: Caution should be taken for using hepatocellular carcinoma cell lines. Our results should be helpful to select proper hepatocellular carcinoma cell lines for biological experiments.

Anti-PD-1 inhibits Foxp3+ Treg cell conversion and unleashes intratumoural effector T cells thereby enhancing the efficacy of a cancer vaccine in a mouse model.

The co-inhibitory molecule PD-1 suppresses T cell responses and has been targeted in the treatment of cancer. Here, we examined the role of PD-1 in regulating the balance between regulatory and effector T cells and whether blocking PD-1 could enhance tumour vaccine-induced protective immunity. A significantly higher proportion of tumour-resident T cells expressed PD-1 and Foxp3 compared with T cells in the tumour circulation or draining lymph nodes, and this correlated with a lower frequency of IFN-γ- and TNF-secreting CD8 T cells. Blocking PD-1 with a specific antibody reduced Foxp3+ regulatory T (Treg) cell induction and enhanced proliferation, cytokine production, and tumour killing by CD8 T cells. Treatment of CT26 tumour-bearing mice with anti-PD-1 in combination with a vaccine, comprising heat-shocked irradiated tumour cells and a TLR 7/8 agonist, significantly reduced tumour growth and enhanced survival. Furthermore, surviving mice resisted tumour re-challenge. The rejection of tumours in mice treated with the anti-PD-1 vaccine combination was associated with a reduction in tumour-infiltrating Treg cells and enhancement of IFN-γ-secreting CD8 T cells. Our findings demonstrate that high PD-1 expression correlates with increased tumour-infiltrating Treg cells and reduced effector T cells and that when combined with a potent antigen-adjuvant combination, blocking PD-1 effectively enhances anti-tumour immunity.

Influences of Allee effects in the spreading of malignant tumours.

A recent study by Korolev et al. [Nat. Rev. Cancer, 14:371-379, 2014] evidences that the Allee effect-in its strong form, the requirement of a minimum density for cell growth-is important in the spreading of cancerous tumours. We present one of the first mathematical models of tumour invasion that incorporates the Allee effect. Based on analysis of the existence of travelling wave solutions to this model, we argue that it is an improvement on previous models of its kind. We show that, with the strong Allee effect, the model admits biologically relevant travelling wave solutions, with well-defined edges. Furthermore, we uncover an experimentally observed biphasic relationship between the invasion speed of the tumour and the background extracellular matrix density.

Magnetic sentinel lymph node biopsy in a murine tumour model.

The magnetic technique for sentinel node biopsy provides a radioisotope-free alternative for staging breast cancer. It requires refinement to reduce "residual iron content" at injection sites by maximising lymphatic uptake to prevent "void artefacts" on magnetic resonance imaging (MRI), which could adversely affect clinical use. The site and timing of injection of magnetic tracer was evaluated in a murine tumour model (right hind limb) in 24 wild type mice. Right-sided intratumoural and left sided subcutaneous injection of magnetic tracer and assessment of nodal iron uptake on MRI, surgical excision and histopathological grading at time frames up to 24 hours were performed. Rapid iron uptake on MRI, smaller "void artefacts"(P<0.001) and a significant increase in iron content with time were identified in the subcutaneous injection group (r=0.937; P<0.001).Subcutaneous injection and increasing delay between tracer injection and surgery is beneficial for lymphatic iron uptake. FROM THE CLINICAL EDITOR: Sentinel lymph node biopsy (SLNB) has been the standard of care in breast cancer management for some time. Recent development has seen the introduction of magnetic tracer for SLNB. In this article, the authors investigated the refined use of magnetic tracer in determining the optimal timing of administration and the location of injection. The findings should provide more data on the future use of this new technique.

The impact of radio-chemotherapy on tumour cells interaction with optimal control and sensitivity analysis.

Oncologists and applied mathematicians are interested in understanding the dynamics of cancer-immune interactions, mainly due to the unpredictable nature of tumour cell proliferation. In this regard, mathematical modelling offers a promising approach to comprehend this potentially harmful aspect of cancer biology. This paper presents a novel dynamical model that incorporates the interactions between tumour cells, healthy tissue cells, and immune-stimulated cells when subjected to simultaneous chemotherapy and radiotherapy for treatment. We analysed the equilibria and investigated their local stability behaviour. We also study transcritical, saddle-node, and Hopf bifurcations analytically and numerically. We derive the stability and direction conditions for periodic solutions. We identify conditions that lead to chaotic dynamics and rigorously demonstrate the existence of chaos. Furthermore, we formulated an optimal control problem that describes the dynamics of tumour-immune interactions, considering treatments such as radiotherapy and chemotherapy as control parameters. Our goal is to utilize optimal control theory to reduce the cost of radiotherapy and chemotherapy, minimize the harmful effects of medications on the body, and mitigate the burden of cancer cells by maintaining a sufficient population of healthy cells. Cost-effectiveness analysis is employed to identify the most economical strategy for reducing the disease burden. Additionally, we conduct a Latin hypercube sampling-based uncertainty analysis to observe the impact of parameter uncertainties on tumour growth, followed by a sensitivity analysis. Numerical simulations are presented to elucidate how dynamic behaviour of model is influenced by changes in system parameters. The numerical results validate the analytical findings and illustrate that a multi-therapeutic treatment plan can effectively reduce tumour burden within a given time frame of therapeutic intervention.

Auranofin loaded silk fibroin nanoparticles for colorectal cancer treatment.

Colorectal cancer (CRC) is the second most common cause of cancer related deaths worldwide and the prevalence in young people especially is increasing annually. In the search for innovative approaches to treat the disease, drug delivery systems (DDS) are promising owing to their unique properties, which allow improved therapeutic results with lower drug concentrations, overcoming drug resistance and at the same time potentially reducing side effects. Silk fibroin is a biopolymer that can be processed to obtain biocompatible and biodegradable nanoparticles that can be efficiently loaded by surface adsorption with small-molecule therapeutics and allow their transport and sustained release by modulating their pharmacokinetics. Auranofin (AF) has recently been repurposed for its strong anticancer activity and is currently in clinical trials. Its mechanism of action is through the inhibition of thioredoxin reductase enzymes, which play an essential role in several intracellular processes and are overexpressed in some tumours. Taking into account that AF has a low solubility in water, we propose silk fibroin nanoparticles (SFN) as AF carrier in order to improve its bioavailability, increasing cellular absorption and preventing its degradation or avoiding some resistance mechanisms. Here we report the preparation and characterization of a new formulation of AF-loaded silk fibroin nanoparticles (SFN-AF), its functionalization with FITC for the analysis of cellular uptake, as well as its cytotoxic activity against cell lines of human colorectal cancer (HT29 and HCT116) in both 2D and 3D cell cultures. 3D spheroid models provide a 3D environment which mimics the 3D aspects of CRC observed in vivo and represents an effective 3D environment to screen therapeutics for the treatment of CRC. The loaded nanoparticles showed a spherical morphology with a hydrodynamic diameter of ~ 160 nm and good stability in aqueous solution due to their negative surface charges. FESEM-EDX analysis revealed a homogeneous distribution of Au clusters with high electron density on the surface of the nanoparticles. SFN-AF incubated in phosphate buffer at 37 °C released 77% of the loaded AF over 10 days, showing an initial burst and then sustained release. Flow cytometry analysis showed that FITC-SFN-AF was efficiently internalized by both cell lines, which was confirmed by confocal microscopy imaging. SFN enhanced the cytotoxicity of AF in 2D cultures in both CRC lines. Promising results were also obtained in 3D culture paving the way for future application of this strategy as a therapy for CRC.

Fluid-Dynamic Culture of Tumour and Immune Cells for More Predictive Infiltration Studies and Immunotherapy Drug Screening.

Immunotherapies represent one of the current most promising challenges in cancer treatment. They are based on the boost of natural immune responses, aimed at cancer eradication. However, the success of immunotherapeutic approaches strictly depends on the interaction between immune cells and cancer cells. Preclinical drug tests currently available are poor in fully predicting the actual safety and efficacy of immunotherapeutic treatments under development. Indeed, conventional 2D cell culture underrepresents the complexity of the tumour microenvironment, while in vivo animal models lack in mimicking the human immune cell responses. In this context, predictability, reliability, and complete immune compatibility still represent challenges to overcome. For this aim, novel 3D, fully humanized in vitro cancer tissue models have been recently optimized by adopting emerging technologies, such as organ-on-chips (OOC) and 3D cancer cell-laden hydrogels. In particular, a novel multi-in vitro organ (MIVO) OOC platform has been recently adopted to culture 3D clinically relevant size cancer tissues under proper physiological culture conditions to investigate anti-cancer treatments and immune-tumour cell crosstalk.The proposed immune-tumour OOC-based model offers a potential tool for accurately modelling human immune-related diseases and effectively assessing immunotherapy efficacy, finally offering promising experimental approaches for personalized medicine.

Establishing a three-dimensional scaffold model of hepatoblastoma.

Introduction: Emerging technologies such as three-dimensional (3D) cell culture and the generation of biological matrices offer exciting new possibilities in disease modelling and tumour therapy. The paucity of laboratory models for hepatoblastoma (HB), the most prevalent malignant liver tumour in children, has hampered the identification of new treatment options for HB patients. We aimed to establish a reliable 3D testing platform using liver-derived scaffolds and HB cell lines that reflect the heterogeneous biology of the disease so as to allow reproducible preclinical research and drug testing. Methods: In a sequence of physical, chemical and enzymatic decellularisation techniques mouse livers were stripped off all cellular components to obtain a 3D scaffold. HB cell lines were then seeded onto these scaffolds and cultivated for several weeks. Results: Our newly generated biological scaffolds consist of liver-specific extracellular matrix components including collagen IV and fibronectin. A cultivation of HB cell lines on these scaffolds led to the formation of 3D tumour structures by infiltration into the matrix. Analyses of drug response to standard-of-care medication for HB showed reliable reproducibility of our stocked models. Discussion: Our HB models are easy-to-handle, producible at large scale, and can be cryopreserved for ready-to-use on-demand application. Our newly generated 3D HB platform may therefore represent a faithful preclinical model for testing treatment response in precision cancer medicine.

A single targeted gamma-ray irradiation induced an acute modulation of immune cells and related cytokines in EMT6 mouse-bearing tumour model.

BACKGROUND: A complicated interplay between radiation doses, tumour microenvironment (TME), and host immune system is linked to the active participation of immune response. OBJECTIVE: The effects of single targeted 2 Gy and 8 Gy gamma-ray irradiations on the immune cell population (lymphocytes, B-cells, T-cells, neutrophils, eosinophils, and macrophages) in EMT6 mouse-bearing tumour models was investigated. METHODS: The effects of both irradiation doses in early (96 hours) and acute phase (5 to 11 days) post-irradiation on immune parameters were monitored in blood circulation and TME using flow cytometry. Simultaneously, selected cytokines related to immune cells within the TME were measured using multiplex ELISA. RESULTS: A temporary reduction in systemic total white blood count (TWBC) resulted from an early phase (96 hours) of gamma-ray irradiation at 2 Gy and 8 Gy compared to sham control group. No difference was obtained in the acute phase. Neutrophils dominated among other immune cells in TME in sham control group. Eosinophils in TME was significantly increased after 8 Gy treatment in acute phase compared to sham control (p< 0.005). Furthermore, the increment of tumour necrosis (TNF)-α, eotaxin and interleukin (IL)-7 (p< 0.05) in both treatment groups and phases were associated with anti-tumour activities within TME by gamma-ray irradiation. CONCLUSION: The temporary changes in immune cell populations within systemic circulation and TME induced by different doses of gamma-ray irradiation correlated with suppression of several pro-tumorigenic cytokines in mouse-bearing EMT6 tumour models.

In vivo tumour imaging employing regional delivery of novel gallium radiolabelled polymer composites.

BACKGROUND: Understanding the regional vascular delivery of particles to tumour sites is a prerequisite for developing new diagnostic and therapeutic composites for treatment of oncology patients. We describe a novel imageable 67Ga-radiolabelled polymer composite that is biocompatible in an animal tumour model and can be used for preclinical imaging investigations of the transit of different sized particles through arterial networks of normal and tumour-bearing organs. RESULTS: Radiolabelling of polymer microspheres with 67Ga was achieved using a simple mix and wash method, with tannic acid as an immobilising agent. Final in vitro binding yields after autoclaving averaged 94.7%. In vivo stability of the composite was demonstrated in New Zealand white rabbits by intravenous administration, and intrahepatic artery instillations were made in normal and VX2 tumour implanted rabbit livers. Stability of radiolabel was sufficient for rabbit lung and liver imaging over at least 3 hours and 1 hour respectively, with lung retention of radiolabel over 91%, and retention in both normal and VX2 implanted livers of over 95%. SPECT-CT imaging of anaesthetised animals and planar imaging of excised livers showed visible accumulation of radiolabel in tumours. Importantly, microsphere administration and complete liver dispersal was more easily achieved with 8 µm diameter MS than with 30 µm MS, and the smaller microspheres provided more distinct and localised tumour imaging. CONCLUSION: This method of producing 67Ga-radiolabelled polymer microspheres is suitable for SPECT-CT imaging of the regional vascular delivery of microspheres to tumour sites in animal models. Sharper distinction of model tumours from normal liver was obtained with smaller MS, and tumour resolution may be further improved by the use of 68Ga instead of 67Ga, to enable PET imaging.

Characterization of cachexia in the human fibrosarcoma HT-1080 mouse tumour model.

BACKGROUND: Cancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT-1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT-1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms. METHODS: Female severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT-1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined. RESULTS: The HT-1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT-1080 tumour-bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross-sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model. CONCLUSIONS: The subcutaneous HT-1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT-1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

Inflammatory Responses during Tumour Initiation: From Zebrafish Transgenic Models of Cancer to Evidence from Mouse and Man.

The zebrafish is now an important model organism for cancer biology studies and provides unique and complementary opportunities in comparison to the mammalian equivalent. The translucency of zebrafish has allowed in vivo live imaging studies of tumour initiation and progression at the cellular level, providing novel insights into our understanding of cancer. Here we summarise the available transgenic zebrafish tumour models and discuss what we have gleaned from them with respect to cancer inflammation. In particular, we focus on the host inflammatory response towards transformed cells during the pre-neoplastic stage of tumour development. We discuss features of tumour-associated macrophages and neutrophils in mammalian models and present evidence that supports the idea that these inflammatory cells promote early stage tumour development and progression. Direct live imaging of tumour initiation in zebrafish models has shown that the intrinsic inflammation induced by pre-neoplastic cells is tumour promoting. Signals mediating leukocyte recruitment to pre-neoplastic cells in zebrafish correspond to the signals that mediate leukocyte recruitment in mammalian tumours. The activation state of macrophages and neutrophils recruited to pre-neoplastic cells in zebrafish appears to be heterogenous, as seen in mammalian models, which provides an opportunity to study the plasticity of innate immune cells during tumour initiation. Although several potential mechanisms are described that might mediate the trophic function of innate immune cells during tumour initiation in zebrafish, there are several unknowns that are yet to be resolved. Rapid advancement of genetic tools and imaging technologies for zebrafish will facilitate research into the mechanisms that modulate leukocyte function during tumour initiation and identify targets for cancer prevention.

IL2RG-deficient minipigs generated via CRISPR/Cas9 technology support the growth of human melanoma-derived tumours.

OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development. MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice. CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.

Assessment of cytotoxic and genotoxic potentials of a mononuclear Fe(II) Schiff base complex with photocatalytic activity in Trigonella.

BACKGROUND: In recent times, coordination complexes of iron in various oxidation states along with variety of ligand systems have been designed and developed for effective treatment of cancer cells without adversely affecting the normal cell and tissues of various organs. METHODS: In this study, we have evaluated the mechanism of action of a Fe(II) Schiff base complex in the crop plant Trigonella foenum-graecum L. (Fenugreek) as the screening system by using morphological, cytological, biochemical and molecular approaches. Further functional characterization was performed using MCF-7 cell line and solid tumour model for the assessment of anti-tumour activity of the complex. RESULTS: Our results indicate efficiency of the Fe(II) Schiff base complex in the induction of double strand breaks in DNA. Complex treatment clearly induced cytotoxic and genotoxic damage in Trigonella seedlings. The Fe-complex treatment caused cell cycle arrest via the activation of ATM-ATR kinase mediated DNA damage response pathway with the compromised expression of CDK1, CDK2 and CyclinB1 protein in Trigonella seedlings. In cultured MCF-7 cells, the complex induces cytotoxicity and DNA fragmentation through intracellular ROS generation. Fe-complex treatment inhibited tumour growth in solid tumour model with no additional side effects. CONCLUSION: The growth inhibitory and cytotoxic effects of the complex result from activation of DNA damage response along with oxidative stress and cell cycle arrest. GENERAL SIGNIFICANCE: Overall, our results have provided comprehensive information on the mechanism of action and efficacy of a Fe(II) Schiff base complex in higher eukaryotic genomes and indicated its future implications as potential therapeutic agent.