Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.




  Functional Tumour Analyses..

Pancreatic Cancer

Other Cancers
  - Signatures of tumour-specific immune
  - Novel therapy by drug-repositioning
  - Serum-based diagnosis: proteins
  - Identification of tumour suppressors
  - Melanoma tumour-niche formation
  - Microenvironmental communication
  - Serum-based diagnosis: microRNAs
  - Identification of cancer stem cell genes
  - Improving the efficiacy of immunotherapy
  - Influence of peritumoral tissue
  - microRNA-based processes

  - Early promotion of PDAC progression



Molecular signatures associated with tumor-specific immune response in melanoma patients treated with dendritic cell-based immunotherapy

We had previously shown that autologous dendritic cells (DCs) loaded with an allogeneic heat shock conditioned melanoma cell-derived lysate, called TRIMEL, induce T-cell-mediated immune responses in stage IV melanoma patients. Importantly, a positive delayed-type hypersensitivity (DTH) reaction against TRIMEL after vaccination correlated with patients prolonged survival. Furthermore, we observed that DTH reaction was associated with a differential response pattern reflected in the presence of distinct cell subpopulations in peripheral blood. Detected variations in patient responses encouraged molecular studies aimed to identify gene expression profiles induced after vaccination in treated patients, allowing the identification of new molecular predictive markers. Gene expression patterns were analysed globally during vaccination, and some of them confirmed in the total leukocyte population of a representative group of responder and non-responder patients. Seventeen genes overexpressed in responder patients after vaccination respect to non-responders were identified, of which ten were linked to immune responses and five related to cell cycle control and signal transduction. In immunological responder patients, increased protein levels of the chemokine receptor CXCR4 and the Fc-receptor CD32 were observed on cell membranes of CD8+ T and B cells and the monocyte population, respectively, confirming gene expression results. Our study contributes to finding molecular markers associated with clinical outcome and better understanding of clinically relevant immunological responses induced by anti-tumour DC-vaccines.
García et al. (2018) Oncotraget 9, 17014-17027. 

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Melanoma microRNA trafficking controls tumour primary niche formation

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Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion.
Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

Dror et al. (2016) Nature Cell Biol. 18, 1006-1017.  pdf icon

Nature Cell Biology cover page

Expansion of a BDCA1+CD14+ myeloid cell population in melanoma patients may attenuate the efficacy of dendritic cell vaccines.

The tumour microenvironment is characterized by regulatory T cells, type II macrophages, myeloid-derived suppressor cells, and other immunosuppressive cells that promote malignant progression. Here we report the identification of a novel BDCA1(+)CD14(+) population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell-based vaccines, where they suppress CD4(+) T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1(+)CD14(+) cells expressed high levels of the immune checkpoint molecule PD-L1 to hinder T-cell proliferation. While this BDCA1(+)CD14(+) cell population expressed markers of both BDCA1(+) dendritic cells and monocytes, analyses of function, transcriptome, and proteome established their unique nature as exploited by tumours for immune escape. We propose that targeting these cells may improve the efficacy of cancer immunotherapy.
Bakdash et al. (2016) Cancer Res. 76, 4332-4346.  pdf icon


scheme of genome-wide shRNA assay

Scheme of genome-wide shRNA knockdown experiments. Meanwhile, the read-out is done by next-generation sequencing instead of microarray analysis as depicted.
Functional screens by means of lentiviral shRNA libraries

RNA interference (RNAi) has become a popular and important tool for the analysis of gene function. Loss-of-function studies, commonly performed by transfection of short interfering RNAs (siRNAs), have greatly facilitated functional analyses of the human transcriptome. However, there are major downsides to siRNA experiments, most importantly the transient inhibition of gene expression as well as their inefficient transfection into non-dividing cells. Overcoming those limitations, short hairpin RNA (shRNA) expression vectors are available, which stably integrate into a target cell's genome via retro- or lentiviral gene transfer. Intracellular processing of shRNAs results in short duplex RNAs with siRNA-like properties. Viral integration ensures a broad range of infectable target cell types and a stable expression of specific shRNAs, resulting in the permanent reduction of the targeted gene product. Complex shRNA expression libraries allow the targeted knockdown of thousands of different genes in a single experiment.
Using such lentiviral vector shRNA libraries and initially barcode arrays and meanwhile next-generation sequencing analysis for decoding of the pooled RNAi screens, we are able to quantify the abundance of individual shRNAs and thus determine in a complex pool the number of cells infected with an individual shRNA construct. We used the technique to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens, for example, identified synthetic-lethal activities toward combination therapies, defined genes which are required for a stem-cell like phenotype and found tumour suppressor genes by in vivo studies. For more details, see below.
Further studies are under way, both for the elucidation of basic regulative processes associated to cancer and for the identification of pathways that are affected by particular drugs or compounds. In particular, we use the technique for obtaining more detailed information on the functional effects of particularly potentially druggable gene products.
More recently, CRISPR-Cas constructs have been used for similar analyses and other purposes.

Wolf et al. (2014) Oncogene 33, 4273-4278. pdf icon
Fredebohm et al. (2013) J. Cell Sci. 126, 3380-3389. pdf icon

Böttcher et al. (2014) BMC Genomics 15, 158. pdf icon
Böttcher et al. (2010) BMC Genomics 11, 7. pdf icon

Wolf et al. (2013) Breast Cancer Res. 15, R109. pdf icon
Böttcher & Hoheisel (2010) Curr. Genom. 11, 162-167.


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