MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern is associated with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as a critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies.info:eu-repo/semantics/publishedVersio

Expression of receptor activator of NFkB (RANK) drives stemness and resistance to therapy in ER+HER2- breast cancer

© Gomes et al. Copyright: Gomes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The role of RANKL-RANK pathway in progesterone-driven mammary carcinogenesis and triple negative breast cancer tumorigenesis has been well characterized. However, and despite evidences of the existence of RANK-positive hormone receptor (HR)-positive breast tumors, the implication of RANK expression in HR-positive breast cancers has not been addressed before. Here, we report that RANK pathway affects the expression of cell cycle regulators and decreases sensitivity to fulvestrant of estrogen receptor (ER)-positive (ER+)/HER2- breast cancer cells, MCF-7 and T47D. Moreover, RANK overexpressing cells had a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy, but were more invasive in vivo. In silico analysis of the transcriptome of human breast tumors, confirmed the association between RANK expression and stem cell and mesenchymal markers in ER+HER2- tumors. Importantly, exposure of ER+HER2- cells to continuous RANK pathway activation by exogenous RANKL, in vitro and in vivo, induced a negative feedback effect, independent of RANK levels, leading to the downregulation of HR and increased resistance to hormone therapy. These results suggest that ER+HER2- RANK-positive cells may constitute an important reservoir of slow cycling, therapy-resistance cancer cells; and that RANK pathway activation is deleterious in all ER+HER2- breast cancer cells, independently of RANK levels.This project was funded by the research project PTDC/MED-ONC/28636/2017 from Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado. IG is supported by the FCT PhD grant SFRH/BD/139178/2018info:eu-repo/semantics/publishedVersio

Comprehensive genomic profiling of cell-free circulating tumor DNA detects response to Ribociclib plus Letrozole in a patient with metastatic breast cancer

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Analysis of cell-free circulating tumor DNA obtained by liquid biopsy is a non-invasive approach that may provide clinically actionable information when conventional tissue biopsy is inaccessible or infeasible. Here, we followed a patient with hormone receptor-positive and human epidermal growth factor receptor (HER) 2-negative breast cancer who developed bone metastases seven years after mastectomy. We analyzed circulating cell-free DNA (cfDNA) extracted from plasma using high-depth massively parallel sequencing targeting 468 cancer-associated genes, and we identified a clonal hotspot missense mutation in the PIK3CA gene (3:178952085, A > G, H1047R) and amplification of the CCND1 gene. Whole-exome sequencing revealed that both alterations were present in the primary tumor. After treatment with ribociclib plus letrozole, the genetic abnormalities were no longer detected in cfDNA. These results underscore the clinical utility of combining liquid biopsy and comprehensive genomic profiling to monitor treatment response in patients with metastasized breast cancer.This work was supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior, Portugal, through Fundos do Orçamento de Estado to Instituto de Medicina Molecular João Lobo Antunes (LA/P/0082/2020), and FCT/FEDER/POR Lisboa 2020, Programa Operacional Regional de Lisboa, PORTUGAL 2020 (LISBOA-01-0145-FEDER-016394), and FEDER/POR Lisboa 2020-Programa Operacional Regional de Lisboa, PORTUGAL 2020 (Infogene, 045300). C.S. was a recipient of a FCT fellowship (SFRH/BDE/110544/2015). This work was funded in part by the National Institutes of Health (NIH)/National Cancer Institute (NCI) Cancer Center Support Grant (P30 CA008748; MSK). J.S.R-F. and B.W. are funded in part by the NIH/NCI P50 CA247749 01 grant and a Breast Cancer Research Foundation grant. J.S.R.-F. is also funded in part by a Susan G Komen leadership grant, and B.W. by a Cycle for Survival grant.info:eu-repo/semantics/publishedVersio

Avaliação dos mecanismos moleculares de resistência a hipometilantes em Leucemia Mieloide Aguda

CORREDEIRA, Patrícia Isabel Martins - Avaliação dos mecanismos moleculares de resistência a hipometilantes em Leucemia Mieloide Aguda. Coimbra : [s.n.], 2016. Dissertação de Mestrado.A leucemia mieloide aguda (LMA) é uma neoplasia complexa e heterogénea da célula progenitora hematopoiética, cujas características incluem desregulação da diferenciação, proliferação clonal e acumulação na medula óssea de células mieloides imaturas. A leucemia promielocítica aguda é um subtipo raro de LMA em que existe acumulação de promielócitos na medula óssea. A maioria dos casos está associado a uma translocação reciproca t(15;17) (q24.1;q21.2) envolvendo os genes PML/RARα. Apesar dos bons resultados da terapêutica com o ácido all-trans retinóico (ATRA), um indutor da diferenciação, alguns doentes desenvolvem efeitos secundários (ex. síndrome do ATRA) e/ou resistência à terapêutica. Neste sentido, tornou-se necessário o desenvolvimento de novas estratégias terapêuticas. Sabendo que as células tumorais estão geralmente, hipermetiladas na zona do promotor de genes supressores tumorais, o que conduz ao seu silenciamento epigenético, foi relevante a partir desta característica desenvolver novas terapias dirigidas a alvos moleculares. Assim surgiu a terapia epigenética através de moduladores epigenéticos, nomeadamente os agentes hipometilantes e os inibidores das desacetilases das histonas. Os hipometilantes como a 5-azacitidina (AZA) e a 2’-deoxy-5-decitabina (DAC) estão atualmente aprovados para o tratamento de algumas neoplasias hematológicas, incluindo leucemias mieloides agudas e síndromes mielodisplásicas. A eficácia da terapia epigenética depende da capacidade de transporte do fármaco para o tecido alvo, entre outros mecanismos. Alterações na atividade e/ou expressão dos transportadores de influxo e efluxo, e ainda em enzimas de metabolismo como a UDP (uridina difosfato) glucuronosil transferase família 1 membro A1 (UGT1A1), podem influenciar a eficiência farmacológica dos fármacos hipometilantes. O objetivo deste trabalho consistiu em avaliar alguns mecanismos moleculares envolvidos na resistência a moduladores epigenéticos (hipometilantes), em particular o envolvimento dos transportadores de efluxo da família ABC e das proteínas vault, na leucemia mieloide aguda. Para a realização do trabalho experimental foi utilizada uma linha celular de LMA/LPA (sem a translocação PML/RARα), a linha celular HL-60, e uma sublinharesistente ao fármaco azacitidina, as células HL-60AZA, (previamente estabelecida no Laboratório de Oncobiologia e Hematologia (LOH) da Faculdade de Medicina da Universidade de Coimbra). Ambas as linhas celulares foram incubadas com doses crescentes de azacitidina e decitabina durante 72h. A proliferação e viabilidade celulares foram avaliadas através do teste de exclusão azul de tripano e o tipo de morte celular por microscopia ótica (morfologia celular) após coloração das células com May-Grünwald-Giemsa. Os níveis de expressão génica do transportador membranar ABCG2 (gene que codifica a proteína BCRP), assim como do gene da enzima UGT1A1, foram determinados por PCR em tempo real. Os níveis de expressão proteica dos transportadores membranares da família ABC, glicoproteína-P (P-gp), MRP1 e da proteína transportadora nuclear major vault (MVP ou LRP), foram avaliados recorrendo à técnica de citometria de fluxo. Por fim, através da técnica de MS-MLPA (Methylation-Specific Multiplex Ligation-dependent Probe Amplification), analisou-se o perfil de metilação de vários genes supressores tumorais (TP73, MSH6, VHL, RARβ, ESR1, CDKN2A, PAX5, KLLN, MGMGT, PAX6, WT1, CD44, GSTP1, ATM, CADM1, CHF3, BRCA2, RB1, THBS1, PYCARD, CDH13, TP53, BCRA1, STK11, GATA5) nas duas linhas celulares em estudo. Em termos de resultados observou-se um efeito citostático (dependente da concentração do fármaco e da linha celular) e citotóxico (dependente da concentração, do tempo de incubação e da linha celular) induzidos pela azacitidina. Estes efeitos foram mais evidentes na linha HL-60 do que na linha resistente, HL-60AZA. O IC50 da linha HL-60AZA foi de 187,1 μM e o da linha HL-60 de 58,5 μM, o que demonstra que as células HL-60AZA são cerca de 3,2 vezes mais resistentes à azacitidina relativamente às células parentais sensíveis. De igual modo, a decitabina induziu um efeito citostático e citotóxico dependentes da concentração de fármaco, do tempo de incubação e da linha celular, superiores na linha celular sensível, comparativamente à linha resistente. No entanto, a concentração de decitabina necessária para atingir o IC50 foi inferior à da azacitidina. A sublinha celular HL-60AZA (cujo IC50 foi de 5195 μM) revelou ser resistente 110,8 vezes superior, comparativamente à linha celular sensível (cujo IC50 foi de 46,9 μM). No presente trabalho observou-se resistência cruzada à decitabina nas células resistentes à azacitidina, indicando, que em caso de resistência a um análogo de nucleósido, haverá resistência a fármacos da mesma família. 5 Morfologicamente, as células HL-60 incubadas durante 72h com 5 μM de azacitidina apresentam menor razão núcleo-citoplasma, comparativamente à situação controlo e às células HL-60AZA incubadas nas mesmas condições. A avaliação dos perfis de metilação e do número de cópias dos genes estudados, não revelou diferenças entre a linha celular HL-60AZA e a linha parental HL-60. No entanto, nas células resistentes observou-se um aumento estatisticamente significativo dos níveis de expressão proteica dos transportadores da família ABC, glicoproteína-P e MRP1, e de MVP e uma tendência para aumento dos níveis de expressão dos genes ABCG2 e UGT1A1. Em conclusão, os resultados sugerem o envolvimento dos transportadores glicoproteína-P, MRP1 e MVP no desenvolvimento de resistência à azacitidina na leucemia mieloide aguda.The acute myeloid leukaemia (AML) is a complex and heterogeneous neoplasia of the hematopoietic parental cell that is characterized by disruption of differentiation, clonal proliferation and bone marrow accumulation of immature myeloid cells. The acute promyelocytic leukaemia is a rare subtype of AML characterised by accumulation of promyelocites in the bone marrow. Most cases are associated with a reciprocal translocation t(15;17) (q21.4;q21.2) involving the PML/RARα genes. Despite the good results of the treatment using all-trans retinoic acid (ATRA), a differentiation inductor, some patients develop side effects (i.e. ATRA syndrome) and/or resistance to therapy. So, novel treatment strategies are deemed as necessary. The knowledge that cancer cells are, generally, hypermethylated in the tumoural suppressor genes’ promotor zone, which causes its epigenetic silencing, was used to develop new therapies with molecular targets. Therefore, the epigenetic therapy through epigenetic modulators has emerged, namely using hypomethylation agents and histone deacetylase inhibitors. Hypomethylants like 5-azacitidine (AZA) and 2’-deoxy-5-decitabine (DAC) are currently approved for treatment of haematological neoplasia, including acute myeloid leukaemia and myelodysplastic syndrome. Epigenetic therapy efficiency depends on the efficacy of transport of the pharmaceuticals into the intended tissue, among other factors. Changes in the activity and/or expression of influx/outflux transporters, and also metabolism enzymes like UPD (uridine diphosphate) glucuronosyltransferase family 1 member A1 (UGT1A1), can influence the pharmaceutical efficiency of hypomethylant pharmaceuticals. The objective of the present work was to evaluate the mechanisms responsible for molecular resistance to epigenetic modulators (hypomethylants), in particular the evolvement of outflux transporters of the ABC family and the vault proteins, in acute myeloid leukaemia (AML). In this study there were used cell lines of AML/APL (without the PML/RARα), HL-60 cell line and azacitidine-resistant cell line (HL-60AZA; previously developed at the Hematology and Oncobiology Laboratory (HOL), Faculty of Medicine, University of Coimbra. Both cell lines were incubated with increasing dosages of azacitidine and decitabine in a period of 72h. The proliferation 8 and cell viability were evaluated using the trypan blue exclusion assay and the type of celular death was evaluated through optical microscopy (cell morphology) after coloration via May-Grünwald-Giemsa dyeing. Expression levels of membrane transporters ABCG2 (gene that transcodes the BCRP protein), and of the metabolism enzyme UGT1A1 were determined through real time PCR. The protein expression levels of the membrane transporters of the ABC family, P-glycoprotein (P-gp), MRP1 and the major vault nuclear transporter (MVP, also known as LPR) were determined using flow cytometry. Finally, the MS-PLPA (Methylation-Specific Multiplex Ligation-dependent Probe Amplification) technique was used to evaluate the methylation profile of the various tumour suppressor genes (TP73, MSH6, VHL, RARβ, ESR1, CDKN2A, PAX5, KLLN, MGMGT, PAX6, WT1, CD44, GSTP1, ATM, CADM1, CHF3, BRCA2, RB1, THBS1, PYCARD, CDH13, TP53, BCRA1, STK11, GATA5) in the two studied cellular lines. The results show that there was a cytostatic (depending on the drug concentration and the cell line) and cytotoxic (depending on the concentration, the incubation time and the cell line) effect induced by the azacitidine. These conclusions were more evident on the HL-60 cell line rather than the resistant line, HL-60AZA. The IC50 at the HL-60AZA cell line was of 187,1 μM and at the HL-60 line was of 58,5 μM, which demonstrates that the HL-60AZA cells are about 3.2 times more resistant to azacitidine relative to the sensitive parental cell line. There were also observed cytostatic and cytotoxic effects varying on the drug concentration, the incubation time and the cell line, in the decitabine incubations, that were higher in the sensitive line relative to the resistant cell line. However, the decitabine concentration necessary to reach IC50 is inferior to that of the azacitidine. The HL-60AZA (which IC50 was 5195 μM) revealed to be 110.8 times more resistant, when compared to the sensitive cell line (that had an IC50 of 46.9 μM). The present work it can be observed that there was developed cross resistance to the decitabine in the azacitidine resistant cells, which indicates that in case of resistance to an analogous nucleoside, there will be resistance to drugs of the same family. Morphologically, the HL-60 cell line incubated in 72h with 5 μM of azacitidine show an inferior nucleus-cytoplasm structural ratio, when compared with the control line and to the HL-60AZA cell line in the same incubation conditions. As of the methylation profiles and number of copies of the studied genes, there was no 9 difference between the HL-60AZA cell subline and the parental HL-60 line. However, in the resistant cells a statistically relevant increase of the proteic expression levels of the ABC family, P-glycoprotein, MRP1 and MVP transporters was observed, as well as a tendency of increasing the expression levels of the ABCG2 and UGT1A1 genes. In conclusion, the results suggest the evolvement of P-glycoprotein, MRP1 and MVP transporters in the developing of resistance to azacitidine in acute myeloid leukaemia

HER2 Expression in Circulating Tumour Cells Isolated from Metastatic Breast Cancer Patients Using a Size-Based Microfluidic Device

HER2 is a prognostic and predictive biomarker in breast cancer, normally assessed in tumour biopsy and used to guide treatment choices. Circulating tumour cells (CTCs) escape the primary tumour and enter the bloodstream, exhibiting great metastatic potential and representing a real-time snapshot of the tumour burden. Liquid biopsy offers the unique opportunity for low invasive sampling in cancer patients and holds the potential to provide valuable information for the clinical management of cancer patients. This study assesses the performance of the RUBYchip™, a microfluidic system for CTC capture based on cell size and deformability, and compares it with the only FDA-approved technology for CTC enumeration, CellSearch®. After optimising device performance, 30 whole blood samples from metastatic breast cancer patients were processed with both technologies. The expression of HER2 was assessed in isolated CTCs and compared to tissue biopsy. Results show that the RUBYchipTM was able to isolate CTCs with higher efficiency than CellSearch®, up to 10 times more, averaging all samples. An accurate evaluation of different CTC subpopulations, including HER2+ CTCs, was provided. Liquid biopsy through the use of the RUBYchipTM in the clinic can overcome the limitations of histological testing and evaluate HER2 status in patients in real-time, helping to tailor treatment during disease evolution

Circulating tumor cell detection methods in renal cell carcinoma : a systematic review

© 2021 Elsevier B.V. All rights reserved.Circulating tumor cells (CTCs) have a potential role as the missing renal cell carcinoma (RCC) biomarker. However, the available evidence is limited, and detection methods lack standardization, hindering clinical use. We performed a systematic review on CTC enrichment and detection methods, and its role as a biomarker in RCC. Full-text screening identified 54 studies. Reviewed studies showed wide heterogeneity, low evidence level, and high risk of bias. Various CTC detection platforms and molecular markers have been used, but none has proven to be superior. CTC detection and CTC count seem to correlate with staging and survival outcomes, although evidence is inconsistent. CTC research is still in an exploratory phase, particularly in RCC. Further studies are still necessary to achieve a standardization of techniques, molecular markers, CTC definitions, and terminology. This is essential to ascertain the role of CTCs as a biomarker and guide future liquid biopsy research in RCC.info:eu-repo/semantics/publishedVersio

Clinical validation of a size-based microfluidic device for circulating tumor cell isolation and analysis in renal cell carcinoma

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0-27), 101.8 (0-255), and 3.2 (0-10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0-255), and 0.2 (0-1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.info:eu-repo/semantics/publishedVersio