Functional
Genome Analysis (B070)
Deutsches
Krebsforschungszentrum,
Im Neuenheimer Feld 580
D-69120
Heidelberg,
Germany. |
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Functional
Tumour Analyses..
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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.
rr
García et al. (2018) Oncotraget 9,
17014-17027. |
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Melanoma
microRNA trafficking controls tumour primary niche formation
r
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.
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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.
rr
Dror et al. (2016) Nature Cell Biol. 18,
1006-1017. 
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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.
rr
Bakdash et al. (2016) Cancer Res. 76,
4332-4346. |
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Scheme of genome-wide
shRNA knockdown experiments. Meanwhile, the read-out is done by
next-generation sequencing instead of microarray analysis as depicted.
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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.
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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.
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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.
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More recently, CRISPR-Cas constructs have been
used for similar analyses and other purposes.
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Wolf et al. (2014) Oncogene 33, 4273-4278. |
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Fredebohm et al. (2013) J. Cell Sci. 126, 3380-3389. |
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Böttcher et al. (2014) BMC Genomics 15,
158. |
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Böttcher et al. (2010) BMC Genomics 11, 7. |
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Wolf et al. (2013) Breast Cancer Res. 15, R109. |
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Böttcher & Hoheisel (2010) Curr. Genom. 11, 162-167. |
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