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1.
Nature ; 611(7935): 365-373, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36323783

RESUMO

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.


Assuntos
Movimento Celular , Líquido Extracelular , Metástase Neoplásica , Neoplasias , Viscosidade , Animais , Embrião de Galinha , Camundongos , Actinas/metabolismo , Líquido Extracelular/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Trocadores de Sódio-Hidrogênio/metabolismo , Canais de Cátion TRPV , Peixe-Zebra/metabolismo , Metástase Neoplásica/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Via de Sinalização Hippo , Esferoides Celulares/patologia , Complexo 2-3 de Proteínas Relacionadas à Actina , Proteína rhoA de Ligação ao GTP , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pulmão/patologia
2.
Int J Mol Sci ; 23(21)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36362093

RESUMO

Multicellular 3D tumor models are becoming a powerful tool for testing of novel drug products and personalized anticancer therapy. Tumor spheroids, a commonly used 3D multicellular tumor model, more closely reproduce the tumor microenvironment than conventional 2D cell cultures. It should be noted that spheroids can be produced using different techniques, which can be subdivided into scaffold-free (SF) and scaffold-based (SB) methods. However, it remains unclear, to what extent spheroid properties depend on the method of their generation. In this study, we aimed to carry out a head-to-head comparison of drug sensitivity and molecular expression profile in SF and SB spheroids along with a monolayer (2D) cell culture. Here, we produced non-small cell lung cancer (NSCLC) spheroids based on human lung adenocarcinoma cell line A549. Drug sensitivity analysis of the tested cell cultures to five different chemotherapeutics resulted in IC50 (A549-SB) > IC50 (A549-SF) > IC50 (A549-2D) trend. It was found that SF and SB A549 spheroids displayed elevated expression levels of epithelial-to-mesenchymal transition (EMT) markers and proteins associated with drug resistance compared with the monolayer A549 cell culture. Enhanced drug resistance of A549-SB spheroids can be a result of larger diameters and elevated deposition of extracellular matrix (ECM) that impairs drug penetration into spheroids. Thus, the choice of the spheroid production method can influence the properties of the generated 3D cell culture and their drug resistance. This fact should be considered for correct interpretation of drug testing results.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Esferoides Celulares/patologia , Linhagem Celular Tumoral , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Matriz Extracelular/patologia , Resistência a Medicamentos , Expressão Gênica , Microambiente Tumoral
3.
Biomed Mater ; 18(1)2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36270604

RESUMO

The incidence of highly aggressive pancreatic cancer is increasing across the globe and is projected to increase to 18.6% by 2050. The mortality rate for this form of cancer is very high and the 5 y relative survival rate is only about 9%-10%. The 3D pancreatic cancer microenvironment exerts a major influence on the poor survival rate. A key factor is the prevention of the penetration of the chemotherapeutic drugs in the three-dimensional (3D) microenvironment leading to the development of chemoresistance which is a major contributor to the survival rates. Hence,in vitrostudies using 3D cultures represent a better approach to understand the effect of therapeutic formulations on the cancer cells when compared to conventional 2D cultures. In the present study, we have explored three different conditions for the development of a 3D pancreatic tumour spheroid model from MiaPaCa-2 and PanC1 cells cultured for 10 days using Matrigel matrix. This optimized spheroid model was employed to evaluate a multi-functional nanotheranostic system fabricated using chitosan nanoparticles co-encapsulated with the chemotherapeutic agent gemcitabine and gold-capped iron oxide nanoparticles for multimodal imaging. The effect of the single and multiple-dose regimens of the theranostic system on the viability of 3D spheroids formed from the two pancreatic cancer cell lines was studied. It was observed that the 3D tumour spheroids cultured for 10 days exhibited resistance towards free gemcitabine drug, unlike the 2D culture. The administration of the multifunctional nanotheranostic system on alternate days effectively reduced the cancer cell viability after five doses to about 20% when compared with other groups. The repeated doses of the nanotheranostic system were found to be more effective than the single dose. Cell line-based differences in internalization of the carrier was also reflected in their response to the nanocarrier with PanC1 showing better sensitivity to the treatment.In vivostudies revealed that the combination of gemcitabine and magnetic field induced hypothermia produced superior regression in cancer when compared with the chemotherapeutic agent alone by a combination of activating the pro-apoptotic pathway and heat-induced necrosis. Our results reveal that this multi-functional system holds promise to overcome the current challenges to treat pancreatic cancers.


Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Humanos , Nanomedicina Teranóstica , Esferoides Celulares/patologia , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Antineoplásicos/farmacologia , Microambiente Tumoral
4.
ACS Biomater Sci Eng ; 8(11): 4648-4672, 2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36260561

RESUMO

Common models used in breast cancer studies, including two-dimensional (2D) cultures and animal models, do not precisely model all aspects of breast tumors. These models do not well simulate the cell-cell and cell-stromal interactions required for normal tumor growth in the body and lake tumor like microenvironment. Three-dimensional (3D) cell culture models are novel approaches to studying breast cancer. They do not have the restrictions of these conventional models and are able to recapitulate the structural architecture, complexity, and specific function of breast tumors and provide similar in vivo responses to therapeutic regimens. These models can be a link between former traditional 2D culture and in vivo models and are necessary for further studies in cancer. This review attempts to summarize the most common 3D in vitro models used in breast cancer studies, including scaffold-free (spheroid and organoid), scaffold-based, and chip-based models, particularly focused on the basic and translational application of these 3D models in drug screening and the tumor microenvironment in breast cancer.


Assuntos
Neoplasias , Esferoides Celulares , Animais , Esferoides Celulares/patologia , Microambiente Tumoral , Técnicas de Cultura de Células/métodos
5.
Biomed Pharmacother ; 153: 113465, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076577

RESUMO

Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.


Assuntos
Neoplasias Colorretais , Fluoruracila , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Cobre/farmacologia , Cobre/uso terapêutico , Dissulfiram/farmacologia , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Esferoides Celulares/patologia , Microambiente Tumoral
6.
J Vis Exp ; (186)2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-36094283

RESUMO

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.


Assuntos
Neoplasias , Esferoides Celulares , Humanos , Imageamento Tridimensional , Neoplasias/patologia , Sefarose , Esferoides Celulares/patologia
7.
Exp Oncol ; 44(2): 121-125, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35964640

RESUMO

BACKGROUND: Hypoxia has been noted as a key factor for induction and maintenance of cancer stemness thereby leading to therapy resistance. Three-dimensional (3D) spheroid models demonstrate a heterogeneity of hypoxic regions replicating the in vivo situation within tumors. Utilizing an established 3D spheroid model, we investigated whether extrinsic hypoxia reinforced chemoresistance in malignant pleural mesothelioma (MPM) spheroids. MATERIALS AND METHODS: Tumor spheres were generated from Meso-1 (a typical human MPM cell line) cells having high spheroid-forming ability. To induce hypoxia condition, we utilized a hypoxia chamber with regulation of O2 and CO2 levels. Cell viability was estimated by a WST-8 assay. Real-time polymerase chain reaction and Western blot were performed to evaluate the expression at mRNA and protein levels. RESULTS: Compared with cells cultured in the two-dimensional monolayer model, tumor sphere cells showed elevated mRNA levels of cancer stemness markers (CD26, CD44 and ABCG2) and protein levels of the stemness and hypoxia adaptation markers (ABCG2, ALDH1A1 and HIFs). Correlating with this, 3D spheroid cells were more resistant to permetrexed and topotecan than the two-dimensional cells, indicative of their potential for hypoxic adaptation. Furthermore, significantly stronger resistance to both chemotherapeutic agents was observed in spheroid cells upon hypoxic challenge compared to spheroid cells under normoxia. CONCLUSION: From the present data, it is concluded that hypoxia adaptation of MPM cells from tumor spheres could enhance their chemoresistance.


Assuntos
Mesotelioma Maligno , Mesotelioma , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Hipóxia/metabolismo , Mesotelioma/tratamento farmacológico , Mesotelioma/genética , Mesotelioma/patologia , RNA Mensageiro , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
8.
J Biomech ; 141: 111229, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35933917

RESUMO

Spheroids are multicellular systems with an interesting rheology giving rise to elasto-visco-plastic properties. They are good tumor models, but the role of the extracellular matrix (ECM) is not fully understood. ECM is an important link between cells and may have a significant impact on tissue organization. Here we determine viscoelastic properties of spheroids including different collagen I amounts using AFM and predict new frequency-dependent properties leading to soft glassy rheology behavior. A unified model - similar to single cell behavior - is proposed and discussed, while complementary confocal experiments reveal the microstructure of spheroids, with collagen I fibers serving as a skeleton for cells, thus reinforcing the spheroid viscoelastic behavior.


Assuntos
Neoplasias , Esferoides Celulares , Colágeno/análise , Colágeno Tipo I , Matriz Extracelular/patologia , Neoplasias/patologia , Reologia , Esferoides Celulares/patologia
9.
Anticancer Res ; 42(8): 3993-4001, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35896235

RESUMO

BACKGROUND/AIM: In a screen of compounds to selectively suppress the growth of cancer spheroids, which contained mutant (mt) KRAS, NPD10621 was discovered and associated derivatives were investigated. MATERIALS AND METHODS: Spheroid areas from HCT116-derived HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were treated with 12 NPD10621 derivatives and measured in three-dimensional floating (3DF) cultures. Several cancers were treated with NPD1018 (pyra-metho-carnil: PMC) in 3DF cultures. In a nude mouse assay, 50% cell growth inhibition (GI50) values were determined. RESULTS: From these 12 derivatives, PMC was the most effective inhibitor of HKe3-mtKRAS spheroid growth with the least toxicity. Furthermore, PMC-mediated growth suppression was observed in all tested cancer cell lines, independent of tissue context, driver gene mutations, and drug resistance, suggesting that the PMC target(s) was crucial for cancer growth in a context-independent manner. The GI50 value of PMC in nude mice assay was 7.7 mg/kg and nude mice that were administered 40 mg/kg PMC for 7 days did not show any abnormal blood cell count values. CONCLUSION: PMC is a low-toxicity compound that inhibits the growth of different tumor cell types.


Assuntos
Neoplasias Colorretais , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Esferoides Celulares/patologia
10.
Int J Mol Sci ; 23(13)2022 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-35806095

RESUMO

In this study, we proposed an in vitro tumor model to simulate the mechanical microenvironment and investigate the effect of compressive stress on the invasion process of malignant tumors. It has been pointed out that the biomechanical environment, as well as the biochemical environment, could affect the transformation of cancer cell migration, invasion, and metastasis. We hypothesized that the solid stress caused by the exclusion of surrounding tissue could transform tumor cells from noninvasive to invasive phenotypes. Colorectal cell spheroids were embedded and cultured in agarose gels of varying concentrations to simulate the earliest stages of tumor formation and invasion. The spheroids embedded in gels at higher concentrations showed peculiar growth after 72 h of culture, and the external compressive loading imposed on them caused peculiar growth even in the gels at lower concentrations. In conclusion, the mechanical microenvironment caused the transformation of tumor cell phenotypes, promoting the growth and invasion of tumor cell spheroids.


Assuntos
Neoplasias , Microambiente Tumoral , Linhagem Celular Tumoral , Movimento Celular , Humanos , Neoplasias/patologia , Esferoides Celulares/patologia
11.
In Vitro Cell Dev Biol Anim ; 58(5): 349-364, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35536385

RESUMO

Tumor development studies should adapt to cancer cells' specific mechanisms in connection with their microenvironment. Standard two-dimensional cultures and gas composition are not relevant to the real cancer environment. Existing three-dimensional models are often requiring sophisticated conditions. Here, we propose and characterize, in two cancer models, melanoma (B16F10) and kidney cancer (RenCa), a three-dimensional culture method, reporting the presence of hypoxia-related genes/proteins and aggressiveness mechanisms (epithelial mesenchymal transition and cancer stem cells). We validate the designed three-dimensional method by comparing it with in vivo growing tumors. The developed method brings simplicity and data reproducibility. Melanoma spheroid-growing cells reached a cell cycle arrest at the G0/G1 phase and showed induction of hypoxia. Spheroid-recovered RenCa cells were enriched in proliferating cells and displayed delayed hypoxia. Moreover, the responses to hypoxia observed in spheroids were validated by in vivo tumor studies for both lines. Three-dimensional shapes induced cancer stem cells in renal cancer, whereas epithelial to mesenchymal transition occurred in the melanoma model. Such distinction in the use of different aggressiveness-leading pathways was observed in in vivo melanoma vs kidney tumors. Thus, this 3D culture model approach is adequate to uncover crucial molecular pathways using distinct mechanisms to reach aggressiveness; i.e., B16F10 cells perform epithelial to mesenchymal transition while RenCa cells dedifferentiate into cancer stem cells. Such three-dimensional models help mimic the in vivo tumor features, i.e., hypoxia and aggressiveness mechanisms as validated here by next-generation sequencing analysis, and are proposed for further alternative methods to in vivo studies.


Assuntos
Neoplasias Renais , Melanoma , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Hipóxia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Melanoma/genética , Melanoma/patologia , Reprodutibilidade dos Testes , Esferoides Celulares/patologia , Microambiente Tumoral
12.
Biomater Sci ; 10(12): 3236-3244, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35579408

RESUMO

iRGD can significantly improve the tumor accumulation and tumor penetration of nanomaterials. However, it still remains unclear how far iRGD can enhance the properties of nanomaterials when its conjugation density is maximized. Herein, we synthesized three types of cylindrical polymer brushes (CPBs) with 0%, 50% and 100% of side chains terminated by iRGD, which were named CPBs-1, CPBs-2 and CPBs-3, respectively, and studied the effects of iRGD density on their cellular uptake, and tumor targeting ability and tumor permeability. It was demonstrated that compared with the iRGD-free CPBs-1, the cellular uptake of CPBs-3 was enhanced 5 times and their tumor accumulation was enhanced twice. The penetration depth of CPBs-3 in three-dimensional multicellular spheroids was larger than 100 µm. Our results provide useful information for the design of active tumor targeting nanomaterials as therapeutics or contrast agents.


Assuntos
Nanoestruturas , Neoplasias , Linhagem Celular Tumoral , Humanos , Neoplasias/patologia , Oligopeptídeos/química , Polímeros/química , Esferoides Celulares/patologia
13.
Methods Mol Biol ; 2429: 555-565, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507189

RESUMO

Cancer stem cells (CSCs) are responsible for the initiation of primary tumors and for metastasis seeding at distant organs. Therefore, they represent crucial targets for the study and preclinical testing of new antimetastatic approaches. We recently generated a molecularly characterized biobank of colorectal CSCs, isolated from individual patients and cultured in serum-free medium as multicellular spheroids. Here, we describe in detail the generation of a metastatic model of colorectal cancer based on the orthotopic injection of CSCs into the cecum serosa of immunodeficient mice. Such a model represents an excellent experimental system to investigate the cellular and molecular mechanisms involved in colorectal cancer metastasis, to analyze rare premetastatic elements such as circulating and disseminated tumor cells, and for the preclinical testing of new agents with potential antimetastatic activity.


Assuntos
Neoplasias Colorretais , Células-Tronco Neoplásicas , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Xenoenxertos , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia
14.
J R Soc Interface ; 19(189): 20210903, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35382573

RESUMO

In vitro tumour spheroids have been used to study avascular tumour growth and drug design for over 50 years. Tumour spheroids exhibit heterogeneity within the growing population that is thought to be related to spatial and temporal differences in nutrient availability. The recent development of real-time fluorescent cell cycle imaging allows us to identify the position and cell cycle status of individual cells within the growing spheroid, giving rise to the notion of a four-dimensional (4D) tumour spheroid. We develop the first stochastic individual-based model (IBM) of a 4D tumour spheroid and show that IBM simulation data compares well with experimental data using a primary human melanoma cell line. The IBM provides quantitative information about nutrient availability within the spheroid, which is important because it is difficult to measure these data experimentally.


Assuntos
Melanoma , Esferoides Celulares , Ciclo Celular , Divisão Celular , Humanos , Melanoma/patologia , Modelos Biológicos , Esferoides Celulares/patologia
15.
Analyst ; 147(10): 2023-2034, 2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35485712

RESUMO

Three-dimensional (3D) multicellular spheroid models can recapitulate the human tumour microenvironment with more accuracy than conventional cell culture models, as they include complex architectural structures and dynamic cellular interactions. Among the diverse platforms for spheroid formation, microfluidic platforms have been extensively applied to study spheroids because they can mimic the in vivo microenvironment. This review provides an overview of the advantages of 3D spheroid cultures with a summary of the recent applications for tumour microenvironment-focused cellular interactions, as well as the studies on spheroids and external stimuli. These 3D tumour spheroid-based microfluidic devices will provide a platform for a better understanding of cellular and external interactions, as well as the discovery of cancer therapeutics.


Assuntos
Neoplasias , Microambiente Tumoral , Técnicas de Cultura de Células , Humanos , Microfluídica , Neoplasias/patologia , Esferoides Celulares/patologia
16.
Int J Mol Sci ; 23(3)2022 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-35163489

RESUMO

Gallbladder cancer (GBC) is one of the most common and aggressive biliary tract cancers with a dismal prognosis. Ongoing clinical trials are evaluating a few selected immune checkpoint inhibitors (ICIs) as monotherapy for the treatment of GBC patients. However, only a subset of patients benefits from these treatments. To improve ICI therapy response, molecular mechanisms that confer resistance to immune checkpoint (IC) blockade needs to be explored. Epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs) have been implicated as key processes that confer ICI treatment resistance. However, in GBC the EMT-CSC-IC axis has not yet been clearly elucidated. This study aims to examine the aberrant expression of ICs associated with CSC and EMT. We successfully enriched CSCs by utilizing a 3-dimensional culture system and established a reversible EMT model with human GBC NOZ cell line. Notably, ICs CD73 and PD-L1 were closely associated with both CSC and EMT phenotypes. Knockdown of CD73 or PD-L1 reduced the proliferative and motile abilities of both adherent monolayers and anchorage-free spheroids. In conclusion, blocking CD73 and PD-L1 offer a promising therapeutic strategy for targeting highly aggressive populations with CSC and EMT phenotype to improve GBC patient prognosis.


Assuntos
5'-Nucleotidase/metabolismo , Antígeno B7-H1/metabolismo , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/metabolismo , Terapia de Alvo Molecular , Biomarcadores Tumorais/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Proteínas Ligadas por GPI/metabolismo , Neoplasias da Vesícula Biliar/imunologia , Neoplasias da Vesícula Biliar/patologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Checkpoint Imunológico/metabolismo , Fatores Imunológicos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fator de Crescimento Transformador beta1/metabolismo , Cicatrização
17.
Int J Mol Sci ; 23(3)2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35163092

RESUMO

2D culture as a model for drug testing often turns to be clinically futile. Therefore, 3D cultures (3Ds) show potential to better model responses to drugs observed in vivo. In preliminary studies, using melanoma (B16F10) and renal (RenCa) cancer, we confirmed that 3Ds better mimics the tumor microenvironment. Here, we evaluated how the proposed 3D mode of culture affects tumor cell susceptibility to anti-cancer drugs, which have distinct mechanisms of action (everolimus, doxorubicin, cisplatin). Melanoma spheroids showed higher resistance to all used drugs, as compared to 2D. In an RCC model, such modulation was only observed for doxorubicin treatment. As drug distribution was not affected by the 3D shape, we assessed the expression of MDR1 and mTor. Upregulation of MDR1 in RCC spheroids was observed, in contrast to melanoma. In both models, mTor expression was not affected by the 3D cultures. By NGS, 10 genes related with metabolism of xenobiotics by cytochrome p450 were deregulated in renal cancer spheroids; 9 of them were later confirmed in the melanoma model. The differences between 3D models and classical 2D cultures point to the potential to uncover new non-canonical mechanisms to explain drug resistance set by the tumor in its microenvironment.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Esferoides Celulares/efeitos dos fármacos , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Sobrevivência Celular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células Tumorais Cultivadas , Microambiente Tumoral
18.
Cell Rep ; 38(7): 110385, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35172160

RESUMO

Plexiform neurofibromas (pNFs) are developmental tumors that appear in neurofibromatosis type 1 individuals, constituting a major source of morbidity and potentially transforming into a highly metastatic sarcoma (MPNST). pNFs arise after NF1 inactivation in a cell of the neural crest (NC)-Schwann cell (SC) lineage. Here, we develop an iPSC-based NC-SC in vitro differentiation system and construct a lineage expression roadmap for the analysis of different 2D and 3D NF models. The best model consists of generating heterotypic spheroids (neurofibromaspheres) composed of iPSC-derived differentiating NF1(-/-) SCs and NF1(+/-) pNF-derived fibroblasts (Fbs). Neurofibromaspheres form by maintaining highly proliferative NF1(-/-) cells committed to the NC-SC axis due to SC-SC and SC-Fb interactions, resulting in SC linage cells at different maturation points. Upon engraftment on the mouse sciatic nerve, neurofibromaspheres consistently generate human NF-like tumors. Analysis of expression roadmap genes in human pNF single-cell RNA-seq data uncovers the presence of SC subpopulations at distinct differentiation states.


Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Neurofibroma Plexiforme/patologia , Células de Schwann/patologia , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Criança , Feminino , Humanos , Masculino , Mesoderma/patologia , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Crista Neural/patologia , Nervo Isquiático/patologia , Esferoides Celulares/patologia , Adulto Jovem
19.
Int J Mol Sci ; 23(4)2022 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-35216378

RESUMO

Despite the enormous progress and development of modern therapies, lung cancer remains one of the most common causes of death among men and women. The key element in the development of new anti-cancer drugs is proper planning of the preclinical research phase. The most adequate basic research exemplary for cancer study are 3D tumor microenvironment in vitro models, which allow us to avoid the use of animal models and ensure replicable culture condition. However, the question tormenting the scientist is how to choose the best tool for tumor microenvironment research, especially for extremely heterogenous lung cancer cases. In the presented review we are focused to explain the key factors of lung cancer biology, its microenvironment, and clinical gaps related to different therapies. The review summarized the most important strategies for in vitro culture models mimicking the tumor-tumor microenvironmental interaction, as well as all advantages and disadvantages were depicted. This knowledge could facilitate the right decision to designate proper pre-clinical in vitro study, based on available analytical tools and technical capabilities, to obtain more reliable and personalized results for faster introduction them into the future clinical trials.


Assuntos
Neoplasias Pulmonares/patologia , Esferoides Celulares/patologia , Microambiente Tumoral/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Humanos , Organoides/patologia
20.
Pathol Int ; 72(3): 176-186, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35147255

RESUMO

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. The spheroid colony formation assay is a useful method to identify cancer stem cells (CSCs). Using the DLD-1 and WiDr CRC cell lines, we performed microarray analyses of spheroid body-forming and parental cells and demonstrated that aldolase, fructose-bisphosphate C (ALDOC) was overexpressed in the spheroid body-forming cells of both lines. Cells transfected with small interfering RNA against ALDOC demonstrated lower proliferation, migration, and invasion compared with negative control cells. Both the number and size of spheres produced by the CRC cells were significantly reduced by ALDOC knockdown. Additionally, inhibition of ALDOC reduced lactate production. Immunohistochemistry was used to analyze ALDOC protein expression in tissues from 135 CRC patients and revealed that 66 (49%) cases were positive for ALDOC. The ALDOC-positive cases were associated with higher T and M grades and, as determined by Kaplan-Meier analysis, a poorer prognosis. Univariate and multivariate analyses indicated that ALDOC expression was an independent prognostic factor for CRC patients. Furthermore, ALDOC expression was associated with CD44 expression. These results suggest that ALDOC contributes to CRC progression and plays an important role in CSCs derived from CRC.


Assuntos
Neoplasias Colorretais/etiologia , Frutose-Bifosfatase/genética , Frutose-Bifosfato Aldolase/genética , Esferoides Celulares/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Frutose-Bifosfatase/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Esferoides Celulares/metabolismo
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