Project description:We performed CITE-seq (10x Genomics-based) to profile and compare the transcriptomes and cell surface expression of immune epitopes in the brains of Cx3cr1+/- and Cx3cr1-/- mice during homeostasis or brain metastasis. We sequenced a total of eight different samples. We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium and later prepare one library consisting of all sight samples and finally separate each sample in silico by it's unique hashing antibody. The samples are as follows: (1) HTO1: brain of naïve Cx3cr1+/- mouse; (2) HTO2: brain of naïve Cx3cr1+/- mouse; (3) HTO3: brain of metastasis-burdened Cx3cr1+/- mouse; (4) HTO4: brain of metastasis-burdened Cx3cr1+/- mouse; (5) HTO5: brain of naïve Cx3cr1-/- mouse; (6) HTO6: brain of naïve Cx3cr1-/- mouse; (7) HTO7: brain of metastasis-burdened Cx3cr1-/- mouse; (8) HTO8: brain of metastasis-burdened Cx3cr1-/- mouse. The following sample comparisons were made: HTO1 and HTO2 versus HTO5 and HTO6; HTO3 and HTO4 versus HTO7 and HTO8.
Project description:We performed CITE-seq (10x Genomics-based) to profile and compare the transcriptomes and cell surface expression of immune epitopes in the brains and blood of different transgenic mice (Cx3cr1CreERT; Cx3cr1CreERT-ROSA-iDTR) during homeostasis or brain metastasis with or without DT-based microglia depletion. We sequenced a total of eight different samples. We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium and later prepare one library consisting of all sight samples and finally separate each sample in silico by it's unique hashing antibody. The samples are as follows: (1) HTO1: brain of naïve Cx3cr1CreERT mouse (non-microglia depleted); (2) HTO2: brain of metastasis-burdened Cx3cr1CreERT mouse (non-microglia depleted); (3) HTO3: brain of metastasis-burdened Cx3cr1CreERT mouse (non-microglia depleted); (4) HTO4: brain of naïve Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted); (5) HTO5: brain of metastasis-burdened Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted); (6) HTO6: brain of metastasis-burdened Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted); (7) HTO7: blood of metastasis-burdened Cx3cr1CreERT mouse (non-microglia depleted); (8) HTO8: blood of metastasis-burdened Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted). The following sample comparisons were made: HTO1 versus HTO4; HTO2 and HTO3 versus HTO5 and HTO6; HTO7 versus HTO8.
Project description:We hypothesized that circulating miRNAs could work as biomarkers for early detection of breast cancer brain metastasis (BCBM), and their targets could constitute new targets for modulation. We used a mouse model of BCBM and Next-Generation Sequencing to establish the circulating miRNAs alterations along brain metastasis development and performed bioinformatics analysis to identify their targets with relevance in the metastatic process. We additionally analyzed human resected brain metastasis of breast cancer patients for target’s expression validation. In our mouse model, we observed a deregulation of circulating miRNAs profile during BCBM progression, with a downregulation of miR-802-5p and miR-194-5p in plasma prior to brain metastases detection. The transcription factor myocyte enhancer factor 2C (MEF2C), was identified as a target for both miRNAs, and its expression was increasingly observed in malignant cells along brain metastasis development. Its upregulation was also observed in peritumoral astrocytes and in human BCBM. Collectively, downregulation of circulating miR-802-5p and miR-194-5p appear as precocious biomarkers for BCBM and MEF2C emerges as a new player and a potential target for modulation.
Project description:Application of a melanoma experimental metastasis model to elucidate molecular mediators of melanoma brain metastasis. Malignant melanoma frequently metastasizes to the brain. The molecular mediators of brain metastasis still remains largely unknnown. Two melanoma cell lines (opposing phenotypes in vitro: invasive/proliferative) were injected (L.V) into immune-compromised animals to generate organ-specific in vivo metastatic tumor cells and host tissue. Immunomagnetic separation was applied to separate tumor cells from host stroma. A rat model was applied to generate organ-specific profiles. Subsequently, a mouse model was applied to generate in vivo brain metastatic samples to follow altered gene expression in melanoma colonizing the brain over time. Gene expression data was collected from human and animal host-specific arrays.
Project description:Breast cancer brain metastasis remains largely incurable. While several mouse models have been developed to investigate the genes and mechanisms regulating breast cancer brain metastasis, these models often lack clinical relevance since they require the use of immune-compromised mice and/or are poorly metastatic to brain from the mammary gland. We describe the development and characterization of an aggressive brain metastatic variant of the 4T1 syngeneic model (4T1Br4) that spontaneously metastasises to lung, bone and brain but is selectively more metastatic to the brain from the mammary gland than parental 4T1 tumors. The 4T1Br4 model will provide a clinically relevant tool to evaluate novel therapies against brain metastasis.
Project description:Brain metastasis is a major complication of lung cancer. An investigation of the pathogenic mechanisms of brain metastasis, as well as the identification of appropriate molecular markers, is necessary. We used microarrays to determine the expression patterns of microRNAs in lung cancer tissue with or without brain metastasis and to investigate the biological role of these miRNAs during tumorigenesis.
Project description:Exclusion of cancer patients with brain metastases from clinical trials is a major cause of the limited therapeutic options available for secondary brain tumors. Here, we report a novel drug-screening platform (METPlatform) based on organotypic cultures that allows identifying anti-metastatic compounds in a preparation that includes the tumor microenvironment. By applying this approach to brain metastasis, we identified HSP90 as a promising therapeutic target. A blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases from melanoma, lung and breast adenocarcinoma with distinct oncogenomic profiles at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis of brain metastases treated with the chaperone inhibitor revealed non-canonical clients of HSP90 as potential novel mediators of brain metastasis and actionable mechanisms of resistance driven by autophagy. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is fully compatible with human samples. We envision that METPlatform could be established as a clinically relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.