| 1. |
- Acero Sanchez, Josep Ll., et al.
(author)
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Electrochemical Genetic Profiling of Single Cancer Cells
- 2017
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:6, s. 3378-3385
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Journal article (peer-reviewed)abstract
- Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.
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| 2. |
- Erickson, A, et al.
(author)
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Spatially resolved clonal copy number alterations in benign and malignant tissue
- 2022
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In: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 608:7922, s. 360-
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Journal article (peer-reviewed)abstract
- Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
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| 3. |
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| 4. |
- Erickson, Andrew, et al.
(author)
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The spatial landscape of clonal somatic mutations in benign and malignant tissue
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Other publication (other academic/artistic)abstract
- Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. Here, we provide an unsupervised approach to study spatial genome integrity in situ to gain molecular insight into clonal relationships. We employed spatially resolved transcriptomics to infer spatial copy number variations in >120 000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
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| 5. |
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| 6. |
- Forsell, Mattias N. E., et al.
(author)
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Regulation of subunit-specific germinal center B cell responses to the HIV-1 envelope glycoproteins by antibody-mediated feedback
- 2017
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In: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 8:JUN
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Journal article (peer-reviewed)abstract
- The regulation of germinal center (GC) B cell responses to single epitopes is well investigated. How monoclonal B cells are regulated within the polyclonal B cell response to protein antigens is less so. Here, we investigate the primary GC B cell response after injection of mice with HIV-1 envelope glycoproteins. We demonstrate that single GCs are seeded by a diverse number of B cell clones shortly after a single immunization and that the presence of Env-specific antibodies can inhibit the development of early GC B cells. Importantly, the suppression was dependent on the GC B cells and the infused antibodies to target the same subunit of the injected HIV-1 envelope glycoproteins. An affinity-dependent antibody feedback has previously been shown to regulate GC B cell development. Here, we propose that this antibody-based feedback acts on GC B cells only if they target the same or overlapping epitopes. This study provides important basic information of GC B cell regulation, and for future vaccine designs with aim to elicit neutralizing antibodies against HIV-1.
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| 7. |
- Kalderén, Christina, et al.
(author)
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CCL2 mediates anti-fibrotic effects in human fibroblasts independently of CCR2
- 2014
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In: International Immunopharmacology. - : Elsevier BV. - 1567-5769 .- 1878-1705. ; 20:1, s. 66-73
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Journal article (peer-reviewed)abstract
- CCL2 is known for its major role as a chemoattractant of monocytes for immunological surveillance and to site of inflammation. CCL2 acts mainly through the G-protein-coupled receptor CCR2 but has also been described to mediate its effects independently of this receptor in vitro and in vivo. Emerging pieces of evidence indicate that the CCL2/CCR2 axis is involved in fibrotic diseases, such as increased plasma levels of CCL2 and the presence of CCL2-hyperresponsive fibroblasts explanted from patients with systemic sclerosis and idiopathic pulmonary fibrosis. One of the profibrotic key mediators is the myofibroblast characterized by overexpression of alpha-smooth muscle actin and collagen I. However, the correlation between the CCL2/CCR2 axis and the activation of fibroblasts is not yet fully understood. We have screened human fibroblasts of various origins, human pulmonary fibroblasts (HPF), human fetal lung fibroblasts (HFL-1) and primary preadipocytes (SPF-1) in regard to CCL2 stimulated fibrotic responses. Surprisingly we found that CCL2 mediates anti-fibrotic effects independently of CCR2 in human fibroblasts of different origins.
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| 8. |
- Kvastad, Linda, et al.
(author)
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Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring
- 2015
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In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 5
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Journal article (peer-reviewed)abstract
- Single cell analysis techniques have great potential in the cancer genomics feld. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.
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| 9. |
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| 10. |
- Kvastad, Linda
(author)
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The Spatial Context – through the lens of method development
- 2021
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Doctoral thesis (other academic/artistic)abstract
- In the present moment of time, we find ourselves in a period where the advancement of genomic tools is progressing at a fast pace. Of particular interest for this thesis is the study of gene activity. What patterns of genes are expressed? Where are they expressed? How can we use this knowledge to improve our quality of life? The research presented in this thesis focuses on developing and applying new tools for interrogating cells and tissues. In Paper I, we describe a protocol for transcript profiling of single cells, capable of measuring the relative expression levels for genes of interest. We successfully applied our method to cancer cells from metastatic breast cancer patients. Profiling 2 to 4 single cells per patient and measuring gene-specific expression from targets previously associated with metastatic breast cancer supports the use of our protocol as a diagnostic tool. In Paper II, we present an assay for spatial RNA quality evaluation, used to estimate the success for tissue specimens before proceeding with more expensive spatial sequencing methods. We showed that the method is capable of measuring high RNA quality in tissue areas of both high and low cell density and that the spatial RNA integrity patterns are reflected in spatial transcriptomics data. In Paper III, we present a protocol for performing spatial mRNA genome-wide expression profiling of FFPE tissue specimens. Thus, we bridge a gap between traditional tissue preservation methods and novel gene technologytools. We found a high Pearson correlation of 0.95 between formalin-fixation paraffin embedding (FFPE) and Fresh Frozen (FF) mouse brain datasets. Although the FPPE samples yielded fewer transcripts and genes compared to FF, there was a high agreement in gene expression between paired anatomical areas for FFPE and FF samples. In Paper IV, we present an approach to investigate in situ transcript derivedinferred copy number variation (iCNV) profiles based on spatial transcriptomics data. In a normal lymph node that displays both distinct gene expression patterns and histological landmarks, we observed a neutral iCNV profile. In contrast, we found huge variabilities investigating several malign tissue types ranging from homogenous (pediatric medulloblastoma) to highly variable genomes (ductal breast cancer and glioblastoma). Strikingly, we also observed similar iCNV profiles in both tumor and benign tissue areas from prostate and skin cancer. In Paper V, we explore the transcriptional and genomic landscape in pediatric tumors from 14 patients. Microglia cells have been implicated to play an important role in the tumor microenvironment, and we found spatial co-localization of microglia and epithelial-to-mesenchymal transition (EMT) signatures in our patient cohort. Furthermore, we found homogenous and recurrent iCNV profiles in the high-grade tumors of relapse patients and identified expression of gene SPP1 in the tumor stroma as a potential prognostic mRNA marker in pediatric brain tumor relapse patients.
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| 11. |
- Kvastad, Linda, et al.
(author)
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The spatial landscape of transcriptomes and genomes in pediatric brain tumors
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Other publication (other academic/artistic)abstract
- Treatment of pediatric brain tumors is continually being improved; still, there is a great need for new treatment options. Here we explore the spatial transcriptomic and genomic landscape in a cohort of pediatric brain tumors using a new generation of unbiased methodologies. We demonstrate the gene expression patterns of the essential cancer-related gene programs of epithelial-to-mesenchymal transition (EMT), the reverse process mesenchymal-to-epithelial transition (MET), and tumor microenvironment (TME) observations through microglia. Furthermore, we identify the gene expression of SPP1 by microglia in the TME as a potential prognostic mRNA marker - in pediatric brain tumor relapse patients.
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| 12. |
- Kvastad, Linda, et al.
(author)
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The spatial RNA integrity number assay for in situ evaluation of transcriptome quality
- 2021
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In: Communications Biology. - : Springer Nature. - 2399-3642. ; 4:1
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Journal article (peer-reviewed)abstract
- The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows. Kvastad et al. develop the spatial RNA Integrity Number (sRIN) assay that evaluates the RNA integrity at cellular resolution. This method improves the resolution of a similar method called the RNA Integrity Number (RIN), demonstrating spatial variation in the quality of RNA samples.
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| 13. |
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| 14. |
- Mirzazadeh, Reza, et al.
(author)
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Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples
- 2023
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Journal article (peer-reviewed)abstract
- Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.
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| 15. |
- Sánchez, J. L. A., et al.
(author)
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Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach
- 2016
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In: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 82, s. 224-232
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Journal article (peer-reviewed)abstract
- Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25 pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".
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| 16. |
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| 17. |
- Villacampa, Eva Gracia, et al.
(author)
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Genome-wide Spatial Expression Profiling in Formalin-fixed Tissues
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Other publication (other academic/artistic)abstract
- Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here we present a procedure to perform genome-wide spatial analysis of mRNA in FFPE fixed tissue sections. The procedure takes advantage of well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3’ end of mRNA molecules in tissue sections. First, we conducted expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method’s capability to delineate anatomical regions from a molecular perspective. Second, we explored the spatial composition of transcriptomic signatures in ovarian carcinosarcoma samples using data-driven analysis methods, exemplifying the method’s potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize organoids and a human lung biopsy specimen infected with SARS-CoV-2.
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| 18. |
- Villacampa, Eva Gracia, et al.
(author)
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Genome-wide spatial expression profiling in formalin-fixed tissues
- 2021
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In: Cell Genomics. - : Elsevier BV. - 2666-979X. ; 1:3
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Journal article (peer-reviewed)abstract
- Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here, we report a procedure to perform genome-wide spatial analysis of mRNA in FFPE-fixed tissue sections, using well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3′ end of mRNA molecules in tissue sections. We applied this method for expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method's capability to delineate anatomical regions from a molecular perspective. We also profiled the spatial composition of transcriptomic signatures in two ovarian carcinosarcoma samples, exemplifying the method's potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize human lung and kidney organoids and a human lung biopsy specimen infected with SARS-CoV-2. We anticipate that genome-wide spatial gene expression profiling in FFPE biospecimens will be used for retrospective analysis of biobank samples, which will facilitate longitudinal studies of biological processes and biomarker discovery.
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