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A key step for metastatic outgrowth involves the generation of a deeply altered microenvironment (niche) that supports the malignant behavior of cancer cells. The complexity of the metastatic niche has posed a significant challenge in elucidating the underlying programs driving its origin. Here, by focusing on early stages of breast cancer metastasis to the lung in mice, we describe a cancer-dependent chromatin remodeling and activation of developmental programs in alveolar type 2 (AT2) cells within the niche. We show that metastatic cells can prime AT2 cells into a reprogrammed multilineage state. In turn, this cancer-induced reprogramming of AT2 cells promoted stem-like features in cancer cells and enhanced their initiation capacity. In conclusion, we propose the concept of "reflected stemness" as an early phenomenon during metastatic niche initiation, wherein metastatic cells reprogram the local tissue into a stem-like state that enhances intrinsic cancer-initiating potential, creating a positive feedback loop where tumorigenic programs are amplified.
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CAR T cells recognizing CD19 effectively treat relapsed and refractory B-ALL and DLBCL. However, CD19 loss is a frequent cause of relapse. Simultaneously targeting a second antigen, CD22, may decrease antigen escape, but is challenging: its density is approximately 10-fold less than CD19, and its large structure may hamper immune synapse formation. The characteristics of the optimal CD22 CAR are underexplored. We generated 12 distinct CD22 antibodies and tested CARs derived from them to identify a CAR based on the novel 9A8 antibody, which was sensitive to low CD22 density and lacked tonic signaling. We found no correlation between affinity or membrane proximity of recognition epitope within Ig domains 3-6 of CD22 with CART function. The optimal strategy for CD19/CD22 CART co-targeting is undetermined. Co-administration of CD19 and CD22 CARs is costly; single CARs targeting CD19 and CD22 are challenging to construct. The co-expression of two CARs has previously been achieved using bicistronic vectors. Here, we generated a dual CART product by co-transduction with 9A8-41BBζ and CAT-41BBζ (obe-cel), the previously described CD19 CAR. CAT/9A8 CART eliminated single- and double-positive target cells in vitro and eliminated CD19- tumors in vivo. CAT/9A8 CART is being tested in a phase I clinical study (NCT02443831).
Assuntos
Linfoma de Burkitt , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T , Recidiva Local de Neoplasia , Imunoterapia Adotiva , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD19 , Anticorpos , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.
Assuntos
Proteoma , Proteômica , Camundongos , Animais , Proteômica/métodos , Glicoproteínas/metabolismo , Açúcares , NucleotídeosRESUMO
MicroRNAs (miRs) are known to regulate pro-inflammatory effector functions of myeloid cells, and miR dysregulation is implicated in rheumatoid arthritis (RA), a condition characterized by inflammation and destruction of the joints. We showed previously that miR-155 is increased in myeloid cells in RA and induces pro-inflammatory activation of monocytes and macrophages; however, its role at the interface between innate and adaptive immunity was not defined. Here, RNA-sequencing revealed that overexpression of miR-155 in healthy donor monocytes conferred a specific gene profile which bears similarities to that of RA synovial fluid-derived CD14+ cells and HLAhighISG15+ synovial tissue macrophages, both of which are characterized by antigen-presenting pathways. In line with this, monocytes in which miR-155 was overexpressed, displayed increased expression of HLA-DR and both co-stimulatory and co-inhibitory molecules, and induced activation of polyfunctional T cells. Together, these data underpin the notion that miR-155-driven myeloid cell activation in the synovium contributes not only to inflammation but may also influence the adaptive immune response.
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Artrite Reumatoide , MicroRNAs , Linfócitos T CD4-Positivos/metabolismo , Humanos , Macrófagos , MicroRNAs/genética , Monócitos , Membrana SinovialRESUMO
INTRODUCTION: Lung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. MATERIALS AND METHODS: A total of 24 pigs were randomized in three groups. Group I (nâ¯=â¯8) underwent sham operation, group II (nâ¯=â¯8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (nâ¯=â¯8) received 3 doses of lazaroid (3â¯mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7â¯tâ¯h postoperative day and lung specimens were harvested for molecular analysis. RESULTS: mRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (-63.7%), LTC4S (-35.9%) and iNOS (-60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). CONCLUSION: Lazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.
Assuntos
Antioxidantes/farmacologia , Arteriopatias Oclusivas/complicações , Lesão Pulmonar/tratamento farmacológico , Pregnatrienos/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Aorta Torácica , Arteriopatias Oclusivas/metabolismo , Modelos Animais de Doenças , Peroxidação de Lipídeos/efeitos dos fármacos , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Masculino , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Oxigênio/síntese química , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , SuínosRESUMO
PURPOSE: The purpose of this study was the development of new quantitative methodologies for the general (total) COL11A1 gene and the C transcript (RT-qPCR methods for A and E transcripts have already been developed by our group previously), the quantification of all COL11A1 transcripts and the investigation for the first time of their potential association with histopathological prognostic factors in lung cancer. METHODS: Real-time RT-qPCR methodologies with dual hybridization probes were developed on the Light Cycler 1.5 platform (Roche,Germany). All COL11A1 transcripts were measured in 27 cDNA lung tissue specimens in a blinded fashion (8 control and 19 non-small cell lung cancer (NSCLC) tissues with known histopathological data). Statistical analysis was performed with the IBM SPSS program. RESULTS: The novel real-time RT-qPCR methodologies were appropriately validated. All 19 NSCLC samples were positive for the general COL11A1 transcript (range 11.2-1198.0 copies/µg total RNA, while 5 out of 8 control samples were negative: mean values were also statistically significantly different (p<0.001). In 4 tumor samples (21%), no specific COL11A1 transcript was detected. Transcript C was detected in only 3 tumor samples. Regarding transcripts A and E, 13 out of 19 tumor samples were positive for either one (68%) and 11 for both (58%). CONCLUSIONS: No other statistically significant association of the specific transcripts with histopathological data was observed, most probably due to the limited number of samples. As the number of general COL11A1 transcripts/µg exceeds the sum of A+E+C transcripts in all samples, there is opportunity for discovery and identification of other transcripts as well.