Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer

Neoplasias genitales; Inmunoterapia; Linfocitos TNeoplàsies genitals; Immunoteràpia; Limfòcits TGenital neoplasms; Immunotherapy; T-LymphocytesBackground Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear. Methods To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+ and CD4+ EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+ and CD4+ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+ and CD4+ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival. Results We found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1− and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+ and PD-1hiCD39+ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hi compared with PD-1− or PD-1dim T cells, and in fact, it was the CD4+PD-1hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+ TILs and PD-1hi and CXCL13 by CD4+ Tconv TILs, correlated with prolonged survival of patients with EC. Conclusions Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hi and CD39 or PD-1hi can be used to select and expand CD8+ and CD4+ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+ and CD4+ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.AG was funded by the Comprehensive Program of Cancer Immunotherapy and Immunology II (CAIMI-II) supported by the BBVA Foundation (grant 53/2021), the La Fundació La Marató de TV3 (201919-30; identification number 488/C/2019), the Spanish Ministry of Science and Innovation (PID2020-118529RB-100) and Instituto de Salud Carlos III (CP15/00058). We thank the CERCA Programme/Generalitat de Catalunya for institutional support. JP was supported by the Beatriu de Pinós programme (BP 2018), cofounded by the Agency for Management of University and Research Grants (AGAUR) and European Union's Horizon 2020. HUB-ICO-IDIBELL Biobank received support from Instituto de Salud Carlos III (PT20/00171) and by Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya (XBTC)

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer

Background: Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear. Methods: To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+ and CD4+ EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+ and CD4+ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+ and CD4+ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival. Results: We found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1- and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+ and PD-1hiCD39+ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hi compared with PD-1- or PD-1dim T cells, and in fact, it was the CD4+PD-1hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+ TILs and PD-1hi and CXCL13 by CD4+ Tconv TILs, correlated with prolonged survival of patients with EC. Conclusions: Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hi and CD39 or PD-1hi can be used to select and expand CD8+ and CD4+ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+ and CD4+ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity

Síntesis y evaluación biológica de agentes antitubulínicos relacionados con colchicina y combretastatina

la tubulina representa un blanco promisorio para las drogas antitumorales debido a su importancia, tanto en elcreci miento de las células tumorales, como de las células endoteliales responsables de la angiogénesis en tumoressó lidos. las combretastatinas, aisladas de la planta Combretum caffrum, inhiben la polimerización de los mi -crotúbu los. Existe un gran interés en sintetizar análogos basados en una de ellas, la combretastatina A-4 (CA-4), preservandoalgunos detalles de su estructura, pero al mismo tiempo mejorando su solubilidad y actividad antitumoral.En este estudio sintetizamos cuatro compuestos relacionados con la combretastatina y evaluamos su efecto sobre laviabilidad, migración y citoesqueleto de líneas tumorales y endoteliales. El compuesto 5a [1-(3,4,5-trimetoxifenil)-6,7-metilendioxi-3,4-dihidroisoquinolina)] inhibió tanto la proliferación como la migración en tres líneas celularesevaluadas. Además, se observó por microscopía confocal, una despolimerización y desintegración de los microtúbulosen las células a concentraciones no-citotóxicas del compuesto 5a (1 μg/ml)

Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs

Comparison of algorithms for the detection of cancer drivers at subgene resolution

Understanding genetic events that lead to cancer initiation and progression remains one of the biggest challenges in cancer biology. Traditionally, most algorithms for cancer-driver identification look for genes that have more mutations than expected from the average background mutation rate. However, there is now a wide variety of methods that look for nonrandom distribution of mutations within proteins as a signal for the driving role of mutations in cancer. Here we classify and review such subgene-resolution algorithms, compare their findings on four distinct cancer data sets from The Cancer Genome Atlas and discuss how predictions from these algorithms can be interpreted in the emerging paradigms that challenge the simple dichotomy between driver and passenger genes.We would like to thank the people working at The Cancer Genome Atlas for their efforts and for making all the data publicly available. E.P.-P. and A.G. acknowledge the support from the Cancer Center grants P30 CA030199 (to our institute) and R35 GM118187 (A.G.). A.K. was supported by startup funds of G.G. and by a collaboration with Bayer AG. D.T. is supported by project SAF2015- 74072-JIN, which is funded by the Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). N.L.-B. acknowledges funding from the European Research Council (consolidator grant 682398). A.V. and T.P. acknowledge funding by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 305444 (RD-Connect).Peer Reviewe

Comparison of algorithms for the detection of cancer drivers at subgene resolution

Understanding genetic events that lead to cancer initiation and progression remains one of the biggest challenges in cancer biology. Traditionally, most algorithms for cancer-driver identification look for genes that have more mutations than expected from the average background mutation rate. However, there is now a wide variety of methods that look for nonrandom distribution of mutations within proteins as a signal for the driving role of mutations in cancer. Here we classify and review such subgene-resolution algorithms, compare their findings on four distinct cancer data sets from The Cancer Genome Atlas and discuss how predictions from these algorithms can be interpreted in the emerging paradigms that challenge the simple dichotomy between driver and passenger genes.E.P.-P. and A.G. acknowledge the support from the Cancer Center grants P30 CA030199 (to our institute) and R35 GM118187 (A.G.). A.K. was supported by startup funds of G.G. and by a collaboration with Bayer AG. D.T. is supported by project SAF2015-74072-JIN, which is funded by the Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). N.L.-B. acknowledges funding from the European Research Council (consolidator grant 682398). A.V. and T.P. acknowledge funding by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 305444 (RD-Connect

Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient’s peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs

Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs

Effect of aging on the human myometrium at single-cell resolution

Abstract Age-associated myometrial dysfunction can prompt complications during pregnancy and labor, which is one of the factors contributing to the 7.8-fold increase in maternal mortality in women over 40. Using single-cell/single-nucleus RNA sequencing and spatial transcriptomics, we have constructed a cellular atlas of the aging myometrium from 186,120 cells across twenty perimenopausal and postmenopausal women. We identify 23 myometrial cell subpopulations, including contractile and venous capillary cells as well as immune-modulated fibroblasts. Myometrial aging leads to fewer contractile capillary cells, a reduced level of ion channel expression in smooth muscle cells, and impaired gene expression in endothelial, smooth muscle, fibroblast, perivascular, and immune cells. We observe altered myometrial cell-to-cell communication as an aging hallmark, which associated with the loss of 25 signaling pathways, including those related to angiogenesis, tissue repair, contractility, immunity, and nervous system regulation. These insights may contribute to a better understanding of the complications faced by older individuals during pregnancy and labor