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In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.
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Aplysia , Arginina , Lisina , Polietilenoglicóis , Animais , Arginina/farmacologia , Arginina/química , Lisina/farmacologia , Lisina/química , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Toxinas Marinhas/farmacologia , Toxinas Marinhas/uso terapêutico , Toxinas Marinhas/química , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , L-Aminoácido Oxidase/farmacologia , L-Aminoácido Oxidase/metabolismo , L-Aminoácido Oxidase/química , Feminino , Linhagem Celular TumoralRESUMO
While various engineering disciplines spent years on developing methods and workflows to increase their R&D efficiency, the field of cell biology has seen limited evolution in the fundamental approaches to interact with living cells. Perturbations are mostly of chemical nature, and physiologically relevant contexts and stimuli are left with limited attention, resulting in a solution space constrained within the boundaries of presently manageable perturbations. To predict in the laboratory how a drug will work in a human patient, cell biology must have a closer look at life and strive to mimic the human being in all his complexity. By implementing an iterative process from perturbation to measurement and vice versa, the authors suggest using a sensor-extended imaging workflow to implement product development practices to cell biology, opening a physiologically relevant solution space for the development of truly translational and predictive fit for purpose in vitro cell models.
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Diagnóstico por Imagem , Software , Humanos , Fluxo de TrabalhoRESUMO
Colorectal cancer (CRC) is one of the deadliest cancers worldwide. The dysregulation of secretory pathways is a crucial driver of CRC progression, since it modulates cell proliferation, angiogenesis and survival. This study explores the changes in the CRC cytokine profile depending on the culture conditions and the presence of fibroblasts and macrophages as cellular components of the tumor microenvironment in 2D and in 3D formed spheroids. Upon analysis of 45 different cytokines, chemokines and growth factors, 20 CRC cell lines were categorized into high and low secretors. In the high secretor group cytokines related to angiogenesis, EMT and invasion were significantly upregulated. LIF and HFG were identified as the best discriminator between both groups. Independent of this grouping, the addition of normal as well as cancer-associated fibroblasts had a similar impact on the cytokine profile by increasing the total amount of secreted cytokines in most of the investigated cell lines. In contrast, the differentiation and polarization of macrophages was modulated differently by normal vs. cancer-associated fibroblasts. In summary, we identified two groups of CRC cell lines that differ in their cytokine profile. The dependance of this profile was analyzed in detail-not only from the tumor cell line but as well from the culture condition in vitro. Key cytokines that discriminate the two groups were identified and their importance as promising biomarker candidates for CRC discussed.
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Neoplasias do Colo , Neoplasias Colorretais , Humanos , Neoplasias Colorretais/patologia , Processos Neoplásicos , Linhagem Celular Tumoral , Citocinas/metabolismo , Microambiente TumoralRESUMO
AIMS: How and why lymphoma cells home to the central nervous system and vitreoretinal compartment in primary diffuse large B-cell lymphoma of the central nervous system remain unknown. Our aim was to create an in vivo model to study lymphoma cell tropism to the central nervous system. METHODS: We established a patient-derived central nervous system lymphoma xenograft mouse model and characterised xenografts derived from four primary and four secondary central nervous system lymphoma patients using immunohistochemistry, flow cytometry and nucleic acid sequencing technology. In reimplantation experiments, we analysed dissemination patterns of orthotopic and heterotopic xenografts and performed RNA sequencing of different involved organs to detect differences at the transcriptome level. RESULTS: We found that xenografted primary central nervous system lymphoma cells home to the central nervous system and eye after intrasplenic transplantation, mimicking central nervous system and primary vitreoretinal lymphoma pathology, respectively. Transcriptomic analysis revealed distinct signatures for lymphoma cells in the brain in comparison to the spleen as well as a small overlap of commonly regulated genes in both primary and secondary central nervous system lymphoma. CONCLUSION: This in vivo tumour model preserves key features of primary and secondary central nervous system lymphoma and can be used to explore critical pathways for the central nervous system and retinal tropism with the goal to find new targets for novel therapeutic approaches.
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Neoplasias do Sistema Nervoso Central , Linfoma Difuso de Grandes Células B , Neoplasias da Retina , Humanos , Animais , Camundongos , Xenoenxertos , Neoplasias da Retina/diagnóstico , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/patologia , Corpo Vítreo/metabolismo , Corpo Vítreo/patologia , Neoplasias do Sistema Nervoso Central/patologia , Sistema Nervoso Central/patologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Retina/metabolismoRESUMO
Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air-liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.
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Fenômenos Biológicos , Neoplasias Ovarianas , Feminino , Humanos , Camundongos , Animais , Perfusão , Microambiente TumoralRESUMO
Background: Primary vitreoretinal lymphoma (PVRL), a rare malignancy of the eye, is strongly related to primary central nervous system lymphoma (PCNSL). We hypothesized that lymphoma cells disseminate to the CNS and eye tissue via distinct homing receptors. The objective of this study was to test expression of CXCR4, CXCR5, CXCR7 and CD44 homing receptors on CD20 positive B-lymphoma cells on enucleated eyes using a PCNSL xenograft mouse model. Methods: We used indirect immunofluorescence double staining for CD20/CXCR4, CD20/CXCR5, CD20/CXCR7 and CD20/CD44 on enucleated eyes of a PCNSL xenograft mouse model with PVRL phenotype (PCNSL group) in comparison to a secondary CNS lymphoma xenograft mouse model (SCNSL group). Lymphoma infiltration was evaluated with an immunoreactive score (IRS). Results: 11/13 paired eyes of the PCNSL but none of the SCNSL group were infiltrated by CD20-positive cells. Particularly the choroid and to a lesser extent the retina of the PCNSL group were infiltrated by CD20+/CXCR4+, CD20+/CXCR5+, few CD20+/CD44+ but no CD20+/CXCR7+ cells. Expression of CXCR4 (p = 0.0205), CXCR5 (p = 0.0004) and CD44 (p < 0.0001) was significantly increased in the PCNSL compared to the SCNSL group. Conclusions: CD20+ PCNSL lymphoma cells infiltrating the eye co-express distinct homing receptors such as CXCR4 and CXCR5 in a PVRL homing mouse model. These receptors may be involved in PVRL homing into the eye.
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Neoplasias do Sistema Nervoso Central , Linfoma , Neoplasias da Retina , Animais , Xenoenxertos , Humanos , Receptores de Hialuronatos , Linfoma/patologia , Camundongos , Receptores CXCR4 , Receptores CXCR5 , Corpo Vítreo/patologiaRESUMO
During the past 15 years, a plethora of innovative 3D in vitro systems has been developed. They offer the possibility of identifying crucial cellular and molecular contributors to the disease by permitting manipulation of each in isolation. However, improvements are needed particularly with respect to the predictivity and validity of those models. The major challenge now is to identify which assay and readout combination(s) best suits the current scientific question(s). A deep understanding of the different platforms along with their pros and cons is a prerequisite to make this decision. This review aims to give an overview of the most prominent systems with a focus on applications, translational relevance and adoption drivers from an industry perspective.
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Neoplasias , HumanosRESUMO
BACKGROUND: The field of cancer immunology is rapidly moving towards innovative therapeutic strategies, resulting in the need for robust and predictive preclinical platforms reflecting the immunological response to cancer. Well characterized preclinical models are essential for the development of predictive biomarkers in the oncology as well as the immune-oncology space. In the current study, gold standard preclinical models are being refined and combined with novel image analysis tools to meet those requirements. METHODS: A panel of 14 non-small cell lung cancer patient-derived xenograft models (NSCLC PDX) was propagated in humanized NOD/Shi-scid/IL-2Rnull mice. The models were comprehensively characterized for relevant phenotypic and molecular features, including flow cytometry, immunohistochemistry, histology, whole exome sequencing and cytokine secretion. RESULTS: Models reflecting hot (>5% tumor-infiltrating lymphocytes/TILs) as opposed to cold tumors (<5% TILs) significantly differed regarding their cytokine profiles, molecular genetic aberrations, stroma content, and programmed cell death ligand-1 status. Treatment experiments including anti cytotoxic T-lymphocyte-associated protein 4, anti-programmed cell death 1 or the combination thereof across all 14 models in the single mouse trial format showed distinctive tumor growth response and spatial immune cell patterns as monitored by computerized analysis of digitized whole-slide images. Image analysis provided for the first time qualitative evaluation of the extent to which PDX models retain the histological features from their original human donors. CONCLUSIONS: Deep phenotyping of PDX models in a humanized setting by combinations of computational pathology, immunohistochemistry, flow cytometry and proteomics enables the exhaustive analysis of innovative preclinical models and paves the way towards the development of translational biomarkers for immuno-oncology drugs.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Citocinas , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Docosahexaenoic acid (DHA) reduces breast cancer tumor growth in preclinical models. To better understand how DHA amplifies the actions of docetaxel (TXT) chemotherapy, we examined the effects of two doses of dietary DHA on tumor size, membrane DHA content and necroptosis using a drug resistant triple negative breast cancer (TNBC) patient derived xenograft (PDX) model. Female NOD.Cb-PrkdcscidIl2rg mice bearing TNBC PDXs were randomized to one of three nutritionally complete diets (20% w/w fat): control (0% DHA), high DHA (3.8% HDHA), or low DHA (1.6% LDHA) with or without intraperitoneal injections of 5 mg/kg TXT, twice weekly for 6 weeks (n=8 per group). Tumors from mice fed either HDHA+TXT or LDHA+TXT were similar in size to each other, but were 36% and 32% smaller than tumors from mice fed control+TXT, respectively (P<.05). A dose effect of DHA incorporation was observed in plasma total phospholipids and in phosphatidylethanolamine and phosphatidylinositol. Both doses of DHA resulted in similarly increased necrotic tissue and decreased NFκB protein expression compared to control tumors, however only the HDHA+TXT had increased expression of necroptosis related proteins: RIPK1, RIPK3 and MLKL (P<.05). Increased MLKL was observed in the lipid raft portion of HDHA+TXT tumor extracts. This work confirms the efficacy of a combination therapy consisting of DHA supplementation and TXT chemotherapy using two doses of DHA as indicated by reduced tumor growth in a TNBC PDX model. Moreover, the results suggest that decreased growth may occur through increased DHA incorporation into tumor phospholipid membranes and necroptosis.
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Ácidos Docosa-Hexaenoicos , Neoplasias de Mama Triplo Negativas , Animais , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/metabolismo , Feminino , Xenoenxertos , Camundongos , Camundongos Endogâmicos NOD , Necroptose , Fosfolipídeos/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: Accurate predictions of tumor dissemination risks and medical treatment outcomes are critical to personalize therapy. Patient-derived xenograft (PDX) models in mice have demonstrated high accuracy in predicting therapeutic outcomes, but methods for predicting tumor invasiveness and early stages of vascular/lymphatic dissemination are still lacking. Here we show that a zebrafish tumor xenograft (ZTX) platform based on implantation of PDX tissue fragments recapitulate both treatment outcome and tumor invasiveness/dissemination in patients, within an assay time of only 3 days. METHODS: Using a panel of 39 non-small cell lung cancer PDX models, we developed a combined mouse-zebrafish PDX platform based on direct implantation of cryopreserved PDX tissue fragments into zebrafish embryos, without the need for pre-culturing or expansion. Clinical proof-of-principle was established by direct implantation of tumor samples from four patients. RESULTS: The resulting ZTX models responded to Erlotinib and Paclitaxel, with similar potency as in mouse-PDX models and the patients themselves, and resistant tumors similarly failed to respond to these drugs in the ZTX system. Drug response was coupled to elevated expression of EGFR, Mdm2, Ptch1 and Tsc1 (Erlotinib), or Nras and Ptch1 (Paclitaxel) and reduced expression of Egfr, Erbb2 and Foxa (Paclitaxel). Importantly, ZTX models retained the invasive phenotypes of the tumors and predicted lymph node involvement of the patients with 91% sensitivity and 62% specificity, which was superior to clinically used tests. The biopsies from all four patient tested implanted successfully, and treatment outcome and dissemination were quantified for all patients in only 3 days. CONCLUSIONS: We conclude that the ZTX platform provide a fast, accurate, and clinically relevant system for evaluation of treatment outcome and invasion/dissemination of PDX models, providing an attractive platform for combined mouse-zebrafish PDX trials and personalized medicine.
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Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Linfonodos/patologia , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
There is a strong rationale for investigating nutritional interventions with docosahexaenoic acid (DHA) in cancer prevention and therapy; however, the effects of DHA on ovarian cancer (OC) have not been well studied. Here, we investigated if DHA alone and in combination with carboplatin reduces OC cell growth in vitro. In vivo, we used a high-grade serous OC patient-derived xenograft (PDX) mouse model to investigate if DHA affects OC growth and enhances the anticancer actions of carboplatin. We showed synergistic cell killing by DHA and carboplatin in DHA-resistant Kuramochi and SKOV3 OC cells, which corresponded with increased DHA incorporation into whole-cell membrane phospholipids (P < 0.05). In vivo, feeding mice a diet supplemented with 3.9% (w/w of fat) DHA resulted in a significant reduction in PDX growth with and without carboplatin (P < 0.05). This reduction in tumor growth was accompanied by an increased tumor necrotic region (P < 0.05) and improved survival. Plasma membranes in tumors and livers excised from mice fed a DHA diet had â¼ twofold increase in DHA incorporation as compared with mice fed a control diet. Our findings indicate that DHA supplementation reduces cancer cell growth and enhances the efficacy of carboplatin in preclinical models of OC through increased apoptosis and necrosis.Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1952453.
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Ácidos Docosa-Hexaenoicos , Neoplasias Ovarianas , Animais , Carboplatina/farmacologia , Carcinoma Epitelial do Ovário , Ciclo Celular , Proliferação de Células , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/farmacologia , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologiaRESUMO
Gamma delta T cells (γδTc) have tremendous anti-tumoral activity, thus γδTc immunotherapy is currently under development for various malignancies. We targeted breast cancer stem-like cells (BCSC), a rare cell population responsible for patient mortality. BCSC were mostly susceptible to γδTc immunotherapy, yet some escaped. The BCSC secretome rendered γδTc hypo-responsive, and resistant BCSC expressed more PD-L1 and anti-apoptotic protein MCL-1 than non-stem-like cells (NSC). BCSC resistance was partially overcome by dMCL1-2, an MCL-1 degrader, or more fully by blocking PD-1 on γδTc. Increased MICA shedding was prevented by the ADAM inhibitor GW280264X, rendering BCSC as sensitive to γδTc cytotoxicity as NSC. Our data show promising potential for γδTc immunotherapy against BCSC while unraveling immune evasion mechanisms exploited by BCSC, which likely also enable their resistance to cytotoxic T and NK cells. Overcoming this resistance, as we have done here, will improve cancer immunotherapy, leading to better cancer patient outcomes.
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Proteínas ADAM/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imunoterapia , Linfócitos Intraepiteliais/imunologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Linfócitos Intraepiteliais/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The immune checkpoint blockade represents a revolution in cancer therapy, with the potential to increase survival for many patients for whom current treatments are not effective. However, response rates to current immune checkpoint inhibitors vary widely between patients and different types of cancer, and the mechanisms underlying these varied responses are poorly understood. Insights into the antitumor activities of checkpoint inhibitors are often obtained using syngeneic mouse models, which provide an in vivo preclinical basis for predicting efficacy in human clinical trials. Efforts to establish in vitro syngeneic mouse equivalents, which could increase throughput and permit real-time evaluation of lymphocyte infiltration and tumor killing, have been hampered by difficulties in recapitulating the tumor microenvironment in laboratory systems. Here, we describe a multiplex in vitro system that overcomes many of the deficiencies seen in current static histocultures, which we applied to the evaluation of checkpoint blockade in tumors derived from syngeneic mouse models. Our system enables both precision-controlled perfusion across biopsied tumor fragments and the introduction of checkpoint-inhibited tumor-infiltrating lymphocytes in a single experiment. Through real-time high-resolution confocal imaging and analytics, we demonstrated excellent correlations between in vivo syngeneic mouse and in vitro tumor biopsy responses to checkpoint inhibitors, suggesting the use of this platform for higher throughput evaluation of checkpoint efficacy as a tool for drug development.
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Inibidores de Checkpoint Imunológico/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Isoenxertos/imunologia , Isoenxertos/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Modelos Biológicos , Receptor de Morte Celular Programada 1/imunologia , Microambiente Tumoral/imunologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0235784.].
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Soft tissue is composed of cells surrounded by an extracellular matrix that is made up of a diverse array of intricately organized proteins. These distinct components work in concert to maintain homeostasis and respond to tissue damage. During tissue repair, extracellular matrix proteins and their degradation products are known to influence physiological processes such as angiogenesis and inflammation. In this study we developed a discovery platform using a decellularized extracellular matrix biomaterial to identify new chemotrophic factors derived from the extracellular matrix. An in vitro culture of RAW.264 macrophage cells with the biomaterial ovine forestomach matrix led to the identification of a novel ~12 kDa chemotactic factor, termed 'MayDay', derived from the N-terminal 31-188 sequence of decorin. The recombinant MayDay protein was shown to be a chemotactic agent for mesenchymal stromal cells in vitro and in vivo. We hypothesize that the macrophage-induced cleavage of decorin, via MMP-12, leads to the release of the chemotactic molecule MayDay, that in turn recruits cells to the site of damaged tissue.
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Fatores Quimiotáticos/farmacologia , Decorina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Fatores Quimiotáticos/química , Quimiotaxia/efeitos dos fármacos , Decorina/química , Células-Tronco Mesenquimais/citologia , Camundongos , Fragmentos de Peptídeos/química , Células RAW 264.7 , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , OvinosRESUMO
Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.
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Regiões 5' não Traduzidas/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Biossíntese de Proteínas/genética , Isoformas de RNA/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Proteína Homeobox Nanog/genética , Proteína Nodal/genética , Fosforilação/efeitos dos fármacos , Fatores de Transcrição da Família Snail/genéticaRESUMO
Rodent models have contributed significantly to the understanding of haematological malignancies. One important model system in this context are patient-derived xenografts (PDX). In the current study, we examined 20 acute leukaemia PDX models for growth behaviour, infiltration in haemopoietic organs and sensitivity towards cytarabine. PDX were injected intratibially (i.t.), intrasplenicaly (i.s.) or subcutaneously (s.c.) into immune compromised mice. For 18/20 models the engraftment capacity was independent of the implantation site. Two models could exclusively be propagated in one or two specific settings. The implantation site did influence tumour growth kinetics as median overall survival differed within one model depending on the injection route. The infiltration pattern was similar in i.t. and i.s. models. In contrast to the s.c. implantation, only one model displayed circulating leukaemic cells outside of the locally growing tumour mass. Cytarabine was active in all four tested models. Nevertheless, the degree of sensitivity was specific for an individual model and implantation site. In summary, all three application routes turned out to be feasible for the propagation of PDX. Nevertheless, the distinct differences between the settings highlight the need for well characterized platforms to ensure the meaningful interpretation of data generated using those powerful tools.
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In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.
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Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
Systemic treatment is necessary for one third of patients with renal cell carcinoma. No valid biomarker is currently available to tailor personalized therapy. In this study we established a representative panel of patient derived xenograft (PDX) mouse models from patients with renal cell carcinomas and determined serum levels of high mobility group B1 (HMGB1) protein under treatment with sunitinib, pazopanib, sorafenib, axitinib, temsirolimus and bevacizumab. Serum HMGB1 levels were significantly higher in a subset of the PDX collection, which exhibited slower tumor growth during subsequent passages than tumors with low HMGB1 serum levels. Pre-treatment PDX serum HMGB1 levels also correlated with response to systemic treatment: PDX models with high HMGB1 levels predicted response to bevacizumab. Taken together, we provide for the first time evidence that the damage associated molecular pattern biomarker HMGB1 can predict response to systemic treatment with bevacizumab. Our data support the future evaluation of HMGB1 as a predictive biomarker for bevacizumab sensitivity in patients with renal cell carcinoma.
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PURPOSE: Although targeting angiogenesis with tyrosine kinase inhibitors (TKIs) has become standard of care in the treatment of clear cell renal cell carcinoma (RCC), resistance mechanism are not fully understood, and there is a need to develop new therapeutic options overcoming them. METHODS AND MATERIALS: To develop a preclinical model that predicts clinical activity of novel agents in 19 RCC patients, we established patient-derived cell (PDC) and xenograft (PDX) models derived from malignant effusions or surgical specimen. RESULTS: Successful PDCs, defined as cells that maintained growth following two passages, were established in 5 of 15 malignant effusions and 1 of 4 surgical specimens. One PDC, clinically refractory to TKIs, was implanted and engrafted in mice, resulting in a comparable histology to the primary tumor. The PDC-PDX model also showed similar genomic features when tested using targeted sequencing of cancer-related genes. When we examined the drug effects of the PDX model, the tumor cells showed resistance to TKIs and everolimus in vitro. CONCLUSION: The results suggest that the PDC-PDX preclinical model we developed using malignant effusions can be a useful preclinical model to interrogate sensitivity to targeted agents based on genomic alterations.