Advancement of precision oncology by integration of highly sensitive protein profiling technologies and patient-derived cell models for functional efficacy testing

2021 ◽  
Vol 2 (4) ◽  
pp. 171-185
Author(s):  
Michael Pawlak ◽  
Markus Templin ◽  
Christian Schmees
Keyword(s):  
Protein Profiling ◽  
Precision Oncology ◽  
Cell Models ◽  
Highly Sensitive

scholarly journals Highly sensitive and multiplexed in situ protein profiling with cleavable fluorescent streptavidin

2019 ◽  
Author(s):  
Renjie Liao ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Jia Guo
Keyword(s):  
Signal Amplification ◽  
Individual Cell ◽  
Protein Detection ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

AbstractThe ability to perform highly sensitive and multiplexed in situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, here we develop an approach using cleavable biotin conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced by at least 10 fold, compared with the existing methods. After imaging, the fluorophores and the biotins unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be unambiguously detected in individual cell in situ.

Abstract 810: Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples

2020 ◽  
Author(s):  
Gerrit Erdmann ◽  
Przemylaw Dudys ◽  
Anja Arndt ◽  
Christian Regenbrecht ◽  
Markus Templin ◽  
...  
Keyword(s):  
Protein Profiling ◽  
Precision Oncology

scholarly journals Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 852 ◽  
Author(s):  
Renjie Liao ◽  
Thai Pham ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Joshua Labaer ◽  
...  
Keyword(s):  
Optical Resolution ◽  
Protein Detection ◽  
Protein Analysis ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

scholarly journals Inferring tumor-specific cancer dependencies through integrating ex-vivo drug response assays and drug-protein profiling

2022 ◽  
Author(s):  
Alina Batzilla ◽  
Junyan Lu ◽  
Jarno Kivioja ◽  
Kerstin Putzker ◽  
Joe Lewis ◽  
...  
Keyword(s):  
Large Scale ◽  
Drug Response ◽  
Ex Vivo ◽  
Cancer Cell Line ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Protein Profiling ◽  
Precision Oncology ◽  
Cancer Therapies ◽  
Her2 Breast Cancer

The development of cancer therapies may be improved by the discovery of tumor-specific molecular dependencies. The requisite tools include genetic and chemical perturbations, each with its strengths and limitations. Drug perturbations can be readily applied to primary cancer samples at a large scale, but mechanistic understanding of hits and further pharmaceutical development is often complicated by the fact that a small compound has a range of affinities to multiple proteins. To computationally infer the molecular dependencies of individual cancers from their ex-vivo drug sensitivity profiles, we developed a mathematical model that deconvolutes these data using measurements of protein-drug affinity profiles. Our method, DepInfeR, correctly identified known dependencies, including EGFR dependence in Her2+ breast cancer cell line, FLT3 dependence in AML tumors with FLT3-ITD mutations, and the differential dependencies on the B-cell receptor pathway in two major subtypes of chronic lymphocytic leukemia (CLL). Furthermore, our method uncovered new subgroup-specific dependencies, including a previously unreported dependence of high-risk CLL on Checkpoint kinase 1 (CHEK1). The method also produced a more accurate map of the molecular dependencies in a heterogeneous set of 117 CLL samples. The ability to deconvolute polypharmacological phenotypes into underlying causal molecular dependencies should increase the utility of high-throughput drug response assays for functional precision oncology.

Ovarian Dysfunction in Fetally Irradiated Beagles

1977 ◽  
Vol 35 ◽  
pp. 658-659
Author(s):  
T. M. Seed ◽  
M. H. Sanderson ◽  
D. L. Gutzeit ◽  
T. E. Fritz ◽  
D. V. Tolle ◽  
...  
Keyword(s):  
Gamma Rays ◽  
Low Dose ◽  
Long Term Effects ◽  
Ovarian Dysfunction ◽  
Dose Rates ◽  
Highly Sensitive ◽  
Microscopic Evaluation ◽  
Ovarian Pathology ◽  
Normal Offspring

The developing mammalian fetus is thought to be highly sensitive to ionizing radiation. However, dose, dose-rate relationships are not well established, especially the long term effects of protracted, low-dose exposure. A previous report (1) has indicated that bred beagle bitches exposed to daily doses of 5 to 35 R 60Co gamma rays throughout gestation can produce viable, seemingly normal offspring. Puppies irradiated in utero are distinguishable from controls only by their smaller size, dental abnormalities, and, in adulthood, by their inability to bear young.We report here our preliminary microscopic evaluation of ovarian pathology in young pups continuously irradiated throughout gestation at daily (22 h/day) dose rates of either 0.4, 1.0, 2.5, or 5.0 R/day of gamma rays from an attenuated 60Co source. Pups from non-irradiated bitches served as controls. Experimental animals were evaluated clinically and hematologically (control + 5.0 R/day pups) at regular intervals.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

Various technic for the measurement of step thickness on crystal surfaces

1978 ◽  
Vol 36 (1) ◽  
pp. 438-439
Author(s):  
C. Boulesteix ◽  
C. Colliex ◽  
C. Mory ◽  
B. Pardo ◽  
D. Renard
Keyword(s):  
Critical Thickness ◽  
Symmetric Case ◽  
Transmitted Intensity ◽  
Crystal Surfaces ◽  
Bright Field ◽  
Excited Wave ◽  
Highly Sensitive ◽  
Contrast Mechanisms ◽  
Step Thickness ◽  
Thickness Variations

Contrast mechanisms, which are responsible of the various types of image formation, are generally thickness dependant. In the following, two imaging modes in the 100 kV CTEM are described : they are highly sensitive to thickness variations and can be used for quantitative estimations of step heights.Detailed calculations (1) of the bright-field intensity have been carried out in the 3 (or 2N+l)-beam symmetric case. They show that in given conditions, the two important symmetric Bloch waves interfere most strongly at a critical thickness for which they have equal emergent amplitudes (the more excited wave at the entrance surface is also the more absorbed). The transmitted intensity I for a Nd2O3 specimen has been calculated as a function of thickness t. The capacity of the method to detect a step and measure its height can be more clearly deduced from a plot of dl/Idt as shown in fig. 1.

Estimation of the noise in TEM image recorded on “imaging plate”

1987 ◽  
Vol 45 ◽  
pp. 748-749
Author(s):  
T. Oikawa ◽  
N. Mori ◽  
T. Katoh ◽  
Y. Harada ◽  
J. Miyahara ◽  
...  
Keyword(s):  
Low Dose ◽  
Quantum Noise ◽  
Laser Light ◽  
Imaging Plate ◽  
Total System ◽  
Electron Dose ◽  
X Ray ◽  
Image Recording ◽  
Recording Material ◽  
Highly Sensitive

The “Imaging Plate”(IP) is a highly sensitive image recording plate for X-ray radiography. It has been ascertained that the IP has superior properties and high practicability as an image recording material in a TEM. The sensitivity, one of the properties, is about 3 orders higher than that of conventional photo film. The IP is expected to be applied to low dose techniques. In this paper, an estimation of the quantum noise on the TEM image which appears in case of low electron dose on the IP is reported.In this experiment, the JEM-2000FX TEM and an IP having the same size as photo film were used.Figure 1 shows the schematic diagram of the total system including the TEM used in this experiment. In the reader, He-Ne laser light is scanned across the IP, then blue light is emitted from the IP.

High-sensitivity detection of gold labels by high-angle dark-field STEM imaging

1990 ◽  
Vol 48 (3) ◽  
pp. 892-893 ◽  
Author(s):  
Max T. Otten
Keyword(s):  
High Sensitivity ◽  
Dark Field ◽  
Bright Field ◽  
Backscattered Electron Imaging ◽  
Backscattered Electrons ◽  
Average Atomic Number ◽  
Highly Sensitive ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
High Sensitivity Detection

Labelling of antibodies with small gold probes is a highly sensitive technique for detecting specific molecules in biological tissue. Larger gold probes are usually well visible in TEM or STEM Bright-Field images of unstained specimens. In stained specimens, however, the contrast of the stain is frequently the same as that of the gold labels, making it virtually impossible to identify the labels, especially when smaller gold labels are used to increase the sensitivity of the immunolabelling technique. TEM or STEM Dark-Field images fare no better (Figs. 1a and 2a), again because of the absence of a clear contrast difference between gold labels and stain.Potentially much more useful is backscattered-electron imaging, since this will show differences in average atomic number which are sufficiently large between the metallic gold and the stains normally used. However, for the thin specimens and at high accelerating voltages of the STEM, the yield of backscattered electrons is very small, resulting in a very weak signal. Consequently, the backscattered-electron signal is often too noisy for detecting small labels, even for large spot sizes.

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