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BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) improves survival in patients with Stage III ovarian cancer following interval cytoreductive surgery (CRS). Optimising patient selection is essential to maximise treatment efficacy and avoid overtreatment. This study aimed to identify biomarkers that predict HIPEC benefit by analysing gene signatures and cellular composition of tumours from participants in the OVHIPEC-1 trial. METHODS: Whole-transcriptome RNA sequencing data were retrieved from high-grade serous ovarian cancer (HGSOC) samples from 147 patients obtained during interval CRS. We performed differential gene expression analysis and applied deconvolution methods to estimate cell-type proportions in bulk mRNA data, validated by histological assessment. We tested the interaction between treatment and potential predictors on progression-free survival using Cox proportional hazards models. RESULTS: While differential gene expression analysis did not yield any predictive biomarkers, the cellular composition, as characterised by deconvolution, indicated that the absence of macrophages and the presence of B cells in the tumour microenvironment are potential predictors of HIPEC benefit. The histological assessment confirmed the predictive value of macrophage absence. CONCLUSION: Immune cell composition, in particular macrophages absence, may predict response to HIPEC in HGSOC and these hypothesis-generating findings warrant further investigation. CLINICAL TRIAL REGISTRATION: NCT00426257.
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Procedimentos Cirúrgicos de Citorredução , Quimioterapia Intraperitoneal Hipertérmica , Neoplasias Ovarianas , Microambiente Tumoral , Humanos , Feminino , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Quimioterapia Intraperitoneal Hipertérmica/métodos , Pessoa de Meia-Idade , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/terapia , Cistadenocarcinoma Seroso/tratamento farmacológico , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Macrófagos/patologia , Macrófagos/metabolismoRESUMO
Complex in vitro models (CIVMs) offer the potential to increase the clinical relevance of preclinical efficacy and toxicity assessments and reduce the reliance on animals in drug development. The European Society of Toxicologic Pathology (ESTP) and Society for Toxicologic Pathology (STP) are collaborating to highlight the role of pathologists in the development and use of CIVM. Pathologists are trained in comparative animal medicine which enhances their understanding of mechanisms of human and animal diseases, thus allowing them to bridge between animal models and humans. This skill set is important for CIVM development, validation, and data interpretation. Ideally, diverse teams of scientists, including engineers, biologists, pathologists, and others, should collaboratively develop and characterize novel CIVM, and collectively assess their precise use cases (context of use). Implementing a morphological CIVM evaluation should be essential in this process. This requires robust histological technique workflows, image analysis techniques, and needs correlation with translational biomarkers. In this review, we demonstrate how such tissue technologies and analytics support the development and use of CIVM for drug efficacy and safety evaluations. We encourage the scientific community to explore similar options for their projects and to engage with health authorities on the use of CIVM in benefit-risk assessment.
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Patologistas , Patologia , Toxicologia , Humanos , Toxicologia/métodos , Animais , Bioengenharia , Testes de Toxicidade , Avaliação Pré-Clínica de Medicamentos , Técnicas In VitroRESUMO
Research on modulation of iodine uptake by thyroid cells could help improve radioiodine treatment of dogs with thyroid tumors. The aim of this study was to characterize the immunohistochemical expression of thyroid transcription factor-1 (TTF-1), thyroglobulin, thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, thyroid peroxidase (TPO), vimentin, and Ki-67 in follicular cell thyroid carcinomas (FTCs) and medullary thyroid carcinomas (MTCs), and to compare protein expression between FTC causing hyperthyroidism and FTC of euthyroid dogs. Immunohistochemistry was performed in 25 FTCs (9 follicular, 8 follicular-compact, and 8 compact) and 8 MTCs. FTCs and MTCs were positive for TTF-1, and expression was higher in FTCs of euthyroid dogs compared with FTCs of hyperthyroid dogs (P= .041). Immunolabeling for thyroglobulin was higher in follicular and follicular-compact FTCs compared with compact FTCs (P = .001), while vimentin expression was higher in follicular-compact FTCs compared with follicular FTCs (P = .011). The expression of TSHR, NIS, pendrin, and TPO was not significantly different among the different subtypes of FTCs or between FTCs causing hyperthyroidism and FTCs in euthyroid dogs. TSHR, NIS, pendrin, and TPO were also expressed in MTCs. Ki-67 labeling index was comparable between FTCs and MTCs, and between FTCs causing hyperthyroidism and FTCs in euthyroid dogs. Proteins of iodine transport were also expressed in canine MTCs, which could have implications for diagnosis and treatment. The different expression of thyroglobulin and vimentin between FTC histological subtypes could reflect variations in tumor differentiation.
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Adenocarcinoma Folicular , Carcinoma Neuroendócrino , Doenças do Cão , Imuno-Histoquímica , Neoplasias da Glândula Tireoide , Cães , Animais , Neoplasias da Glândula Tireoide/veterinária , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Imuno-Histoquímica/veterinária , Carcinoma Neuroendócrino/veterinária , Carcinoma Neuroendócrino/patologia , Carcinoma Neuroendócrino/metabolismo , Adenocarcinoma Folicular/veterinária , Adenocarcinoma Folicular/patologia , Adenocarcinoma Folicular/metabolismo , Tireoglobulina/metabolismo , Masculino , Simportadores/metabolismo , Feminino , Receptores da Tireotropina/metabolismo , Iodeto Peroxidase/metabolismo , Vimentina/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Hipertireoidismo/veterinária , Hipertireoidismo/metabolismo , Hipertireoidismo/patologia , Antígeno Ki-67/metabolismoRESUMO
Thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, and thyroid peroxidase (TPO) are essential for the uptake of iodine by follicular thyroid cells. The aim of this study was to establish immunohistochemistry (IHC) protocols for TSHR, NIS, pendrin, and TPO in canine tissues and characterize their expression in organoids derived from canine follicular cell thyroid carcinoma (FTC) and in the respective primary tumors. This constitutes a fundamental step to establish organoids as a model to study the uptake of iodine in canine FTC. Commercially available antibodies directed against human proteins were selected. Antibody specificity was confirmed by western blot using lysates of the HTori-3 human thyroid cell line and healthy canine thyroid gland. IHC was validated using HTori-3 cells and a set of canine normal tissues including healthy thyroid gland. The expression of TSHR, NIS, pendrin, and TPO was evaluated in 3 organoid lines derived from FTC and respective primary tumors. All 4 antibodies produced specific bands by western blot and cytoplasmic labeling in follicular cells by IHC in both human HTori-3 cells and canine thyroid gland. NIS also showed basolateral membrane immunolabeling in follicular cells. All 4 proteins were highly expressed in organoids derived from FTC. The expression was similar or higher compared to the primary tumors. The results of this study characterize organoids derived from canine FTC as a suitable in vitro model to investigate iodine uptake, opening new research possibilities in the field of canine thyroid cancer therapy.
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Doenças do Cão , Iodo , Neoplasias da Glândula Tireoide , Animais , Cães , Imuno-Histoquímica , Organoides , Neoplasias da Glândula Tireoide/veterináriaRESUMO
Hepatitis C virus (HCV) displays a restricted host species tropism and only humans and chimpanzees are susceptible to infection. A robust immunocompetent animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control, and prophylactic vaccine development. The closest homolog of HCV is the equine nonprimate hepacivirus (NPHV), which shares similar features with HCV and thus represents an animal model to study hepacivirus infections in their natural hosts. We aimed to dissect equine immune responses after experimental NPHV infection and conducted challenge experiments to investigate immune protection against secondary NPHV infections. Horses were i.v. injected with NPHV containing plasma. Flow cytometric analysis was used to monitor immune cell frequencies and activation status. All infected horses became viremic after 1 or 2 wk and viremia could be detected in two horses for several weeks followed by a delayed seroconversion and viral clearance. Histopathological examinations of liver biopsies revealed mild, periportally accentuated infiltrations of lymphocytes, macrophages, and plasma cells with some horses displaying subclinical signs of hepatitis. Following viral challenge, an activation of equine immune responses was observed. Importantly, after a primary NPHV infection, horses were protected against rechallenge with the homologous as well as a distinct isolate with only minute amounts of circulating virus being detectable.
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Hepacivirus/fisiologia , Hepatite C/veterinária , Doenças dos Cavalos/imunologia , Animais , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/imunologia , Hepatite C/prevenção & controle , Hepatite C/virologia , Doenças dos Cavalos/prevenção & controle , Doenças dos Cavalos/virologia , Cavalos , Humanos , Filogenia , Linfócitos T/imunologiaRESUMO
Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.
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Autofagia/genética , Cisteína Endopeptidases/genética , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Vacúolos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Cães , Dados de Sequência Molecular , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/veterinária , Vacúolos/genética , Peixe-ZebraRESUMO
UNLABELLED: Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. IMPORTANCE: The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes.
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Infecções por Bunyaviridae/virologia , Glicoproteínas/genética , Mutação , Orthobunyavirus/patogenicidade , Biossíntese de Proteínas , Proteínas não Estruturais Virais/genética , Proteínas Virais/metabolismo , Animais , Encéfalo/virologia , Linhagem Celular , Genoma Viral , Glicoproteínas/química , Glicoproteínas/metabolismo , Interações Hospedeiro-Patógeno , Interferons/antagonistas & inibidores , Interferons/genética , Camundongos , Orthobunyavirus/química , Orthobunyavirus/genética , Orthobunyavirus/metabolismo , Deleção de Sequência , Carga Viral , Proteínas Virais/genética , Vírion , Fatores de VirulênciaRESUMO
A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Cisteína Endopeptidases/genética , Doenças por Armazenamento dos Lisossomos/veterinária , Doenças Neurodegenerativas/veterinária , Animais , Western Blotting/veterinária , Citoplasma/patologia , Cães , Feminino , Imunofluorescência/veterinária , Imuno-Histoquímica/veterinária , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/patologia , Masculino , Microscopia Eletrônica/veterinária , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Vacúolos/patologiaRESUMO
UNLABELLED: Hepatitis C virus (HCV) has a very narrow species and tissue tropism and efficiently replicates only in humans and the chimpanzee. Recently, several studies identified close relatives to HCV in different animal species. Among these novel viruses, the nonprimate hepaciviruses (NPHV) that infect horses are the closest relatives of HCV described to date. In this study, we analyzed the NPHV prevalence in northern Germany and characterized the clinical course of infection and viral tissue tropism to explore the relevance of HCV-related horse viruses as a model for HCV infection. We found that approximately 31.4% of 433 horses were seropositive for antibodies (Abs) against NPHV and approximately 2.5% carried viral RNA. Liver function analyses revealed no indication for hepatic impairment in 7 of 11 horses. However, serum gamma-glutamyl transferase (GGT) concentrations were mildly elevated in 3 horses, and 1 horse displayed even highly elevated GGT levels. Furthermore, we observed that NPHV infection could be cleared in individual horses with a simultaneous emergence of nonstructural (NS)3-specific Abs and transient elevation of serum levels of liver-specific enzymes indicative for a hepatic inflammation. In other individual horses, chronic infections could be observed with the copresence of viral RNA and NS3-specific Abs for over 6 months. For the determination of viral tissue tropism, we analyzed different organs and tissues of 1 NPHV-positive horse using quantitative real-time polymerase chain reaction and fluorescent in situ hydridization and detected NPHV RNA mainly in the liver and at lower amounts in other organs. CONCLUSION: Similar to HCV infections in humans, this work demonstrates acute and chronic stages of NPHV infection in horses with viral RNA detectable predominantly within the liver.
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Hepacivirus/fisiologia , Hepatite Viral Animal/epidemiologia , Cavalos/virologia , Interações Hospedeiro-Patógeno , Animais , Doença Crônica , Modelos Animais de Doenças , Feminino , Alemanha/epidemiologia , Fígado/virologia , Prevalência , Tropismo ViralRESUMO
Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and "synthetic" SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.
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Infecções por Bunyaviridae/virologia , Córtex Cerebral/virologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Orthobunyavirus/patogenicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Bunyaviridae/imunologia , Infecções por Bunyaviridae/mortalidade , Infecções por Bunyaviridae/patologia , Bovinos , Linhagem Celular , Doenças Cerebelares/imunologia , Doenças Cerebelares/patologia , Doenças Cerebelares/virologia , Córtex Cerebral/imunologia , Córtex Cerebral/patologia , Modelos Animais de Doenças , Progressão da Doença , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Camundongos , Dados de Sequência Molecular , Neurônios/imunologia , Neurônios/patologia , Neurônios/virologia , Orthobunyavirus/genética , Orthobunyavirus/isolamento & purificação , Deleção de Sequência , Ovinos , Medula Espinal/imunologia , Medula Espinal/patologia , Medula Espinal/virologia , Taxa de Sobrevida , Vacúolos , Tropismo Viral , Virulência , Cultura de Vírus , Replicação ViralRESUMO
Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways.
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Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Orthobunyavirus/fisiologia , RNA Polimerase II/metabolismo , Proteínas não Estruturais Virais/metabolismo , Humanos , Orthobunyavirus/imunologia , ProteóliseRESUMO
The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses have been detected in man and various animal species, but, to date, there have been no reports of nidovirus in reptiles. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (>33â000 nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the haemagglutinin-neuraminidase of paramyxoviruses. Analysis of genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity within the order Nidovirales.
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Boidae/virologia , Infecções por Nidovirales/veterinária , Nidovirales/genética , Nidovirales/isolamento & purificação , Pneumonia Viral/veterinária , Animais , Sequência de Bases , Variação Genética , Genoma Viral , Pulmão/patologia , Pulmão/virologia , Dados de Sequência Molecular , Nidovirales/classificação , Infecções por Nidovirales/patologia , Infecções por Nidovirales/virologia , Filogenia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/genética , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/genéticaRESUMO
The consensus molecular subtypes (CMS) of colorectal cancer (CRC) is the most widely-used gene expression-based classification and has contributed to a better understanding of disease heterogeneity and prognosis. Nevertheless, CMS intratumoral heterogeneity restricts its clinical application, stressing the necessity of further characterizing the composition and architecture of CRC. Here, we used Spatial Transcriptomics (ST) in combination with single-cell RNA sequencing (scRNA-seq) to decipher the spatially resolved cellular and molecular composition of CRC. In addition to mapping the intratumoral heterogeneity of CMS and their microenvironment, we identified cell communication events in the tumor-stroma interface of CMS2 carcinomas. This includes tumor growth-inhibiting as well as -activating signals, such as the potential regulation of the ETV4 transcriptional activity by DCN or the PLAU-PLAUR ligand-receptor interaction. Our study illustrates the potential of ST to resolve CRC molecular heterogeneity and thereby help advance personalized therapy.
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Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.
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Núcleo Celular , Perfilação da Expressão Gênica , Ratos , Camundongos , Animais , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Indicadores e Reagentes/metabolismo , Mamíferos/genéticaRESUMO
Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin (VIM), which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis.
Assuntos
Sistemas CRISPR-Cas , Neoplasias Cutâneas , Animais , Cavalos , Oncogenes , Fibroblastos , Marcação de GenesRESUMO
The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM.
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Neoplasias da Mama , Laminina , Animais , Feminino , Humanos , Camundongos , Membrana Basal , Neoplasias da Mama/patologia , Células Endoteliais , Células Epiteliais , Modelos Animais de DoençasRESUMO
Introduction: Tumor-stroma ratio (TSR) is prognostic in multiple cancers, while its role in high-grade serous ovarian cancer (HGSOC) remains unclear. Despite the prognostic insight gained from genetic profiles and tumor-infiltrating lymphocytes (TILs), the prognostic use of histology slides remains limited, while it enables the identification of tumor characteristics via computational pathology reducing scoring time and costs. To address this, this study aimed to assess TSR's prognostic role in HGSOC and its association with TILs. We additionally developed an algorithm, Ovarian-TSR (OTSR), using deep learning for TSR scoring, comparing it to manual scoring. Methods: 340 patients with advanced-stage who underwent primary debulking surgery (PDS) or neo-adjuvant chemotherapy (NACT) with interval debulking (IDS). TSR was assessed in both the most invasive (MI) and whole tumor (WT) regions through manual scoring by pathologists and quantification using OTSR. Patients were categorized as stroma-rich (≥ 50% stroma) or stroma-poor (< 50%). TILs were evaluated via immunohistochemical staining. Results: In PDS, stroma-rich tumors were significantly associated with a more frequent papillary growth pattern (60% vs 34%), while In NACT stroma-rich tumors had a lower Tumor Regression Grading (TRG 4&5, 21% vs 57%) and increased pleural metastasis (25% vs 16%). Stroma-rich patients had significantly shorter overall and progression-free survival compared to stroma-poor (31 versus 45 months; P < 0.0001, and 15 versus 17 months; P = 0.0008, respectively). Combining stromal percentage and TILs led to three distinct survival groups with good (stroma-poor, high TIL), medium (stroma-rich, high TIL, or; stroma-poor, Low TIL), and poor(stroma-rich, low TIL) survival. These survival groups remained significant in CD8 and CD103 in multivariable analysis (Hazard ratio (HR) = 1.42, 95% Confidence-interval (CI) = 1.02-1.99; HR = 1.49, 95% CI = 1.01-2.18, and HR = 1.48, 95% CI = 1.05-2.08; HR = 2.24, 95% CI = 1.55-3.23, respectively). OTSR was able to recapitulate these results and demonstrated high concordance with expert pathologists (correlation = 0.83). Conclusions: TSR is an independent prognostic factor for survival assessment in HGSOC. Stroma-rich tumors have a worse prognosis and, in the case of NACT, a higher likelihood of pleural metastasis. OTSR provides a cost and time-efficient way of determining TSR with high reproducibility and reduced inter-observer variability.
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Although initially successful, treatments with chemotherapy often fail because of the recurrence of chemoresistant metastases. Since these tumors develop after treatment, resistance is generally thought to occur in response to chemotherapy. However, alternative mechanisms of intrinsic chemoresistance in the chemotherapy-naïve setting may exist but remain poorly understood. Here, we study drug-naïve murine breast cancer brain metastases (BCBMs) to identify how cancer cells growing in a secondary site can acquire intrinsic chemoresistance without cytotoxic agent exposure. We demonstrate that drug-naïve murine breast cancer cells that form cancer lesions in the brain undergo vascular mimicry and concomitantly express the adenosine 5'-triphosphate-binding cassette transporter breast cancer resistance protein (BCRP), a common marker of brain endothelial cells. We reveal that expression of BCRP by the BCBM tumor cells protects them against doxorubicin and topotecan. We conclude that BCRP overexpression can cause intrinsic chemoresistance in cancer cells growing in metastatic sites without prior chemotherapy exposure.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Neoplasias da Mama , Animais , Feminino , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células Endoteliais/metabolismo , Proteínas de Neoplasias/metabolismoRESUMO
Mammary tumors in dogs hold great potential as naturally occurring breast cancer models in translational oncology, as they share the same environmental risk factors, key histological features, hormone receptor expression patterns, prognostic factors, and genetic characteristics as their human counterparts. We aimed to develop in vitro tools that allow functional analysis of canine mammary tumors (CMT), as we have a poor understanding of the underlying biology that drives the growth of these heterogeneous tumors. We established the long-term culture of 24 organoid lines from 16 dogs, including organoids derived from normal mammary epithelium or benign lesions. CMT organoids recapitulated key morphological and immunohistological features of the primary tissue from which they were derived, including hormone receptor status. Furthermore, genetic characteristics (driver gene mutations, DNA copy number variations, and single-nucleotide variants) were conserved within tumor-organoid pairs. We show how CMT organoids are a suitable model for in vitro drug assays and can be used to investigate whether specific mutations predict therapy outcomes. Specifically, certain CMT subtypes, such as PIK3CA mutated, estrogen receptor-positive simple carcinomas, can be valuable in setting up a preclinical model highly relevant to human breast cancer research. In addition, we could genetically modify the CMT organoids and use them to perform pooled CRISPR/Cas9 screening, where library representation was accurately maintained. In summary, we present a robust 3D in vitro preclinical model that can be used in translational research, where organoids from normal, benign as well as malignant mammary tissues can be propagated from the same animal to study tumorigenesis.
Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Humanos , Cães , Animais , Feminino , Organoides/metabolismo , Neoplasias da Mama/patologia , Variações do Número de Cópias de DNA , Bancos de Espécimes Biológicos , Neoplasias Mamárias Animais/patologia , Hormônios/metabolismoRESUMO
(1) Background: an increasing number of breast cancer patients develop lethal brain metastases (BM). The complete removal of these tumors by surgery becomes complicated when cells infiltrate into the brain parenchyma. However, little is known about the nature of these invading cells in breast cancer brain metastasis (BCBM). (2) Methods: we use intravital microscopy through a cranial window to study the behavior of invading cells in a mouse model of BCBM. (3) Results: we demonstrate that BCBM cells that escape from the metastatic mass and infiltrate into brain parenchyma undergo epithelial-to-mesenchymal transition (EMT). Moreover, cells undergoing EMT revert to an epithelial state when growing tumor masses in the brain. Lastly, through multiplex immunohistochemistry, we confirm the presence of these infiltrative cells in EMT in patient samples. (4) Conclusions: together, our data identify the critical role of EMT in the invasive behavior of BCBM, which warrants further consideration to target those cells when treating BCBM.