Targeted Expansion Sequencing Protocols v3

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Fei Chen ◽  
Asmamaw T. Wassie ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Pilot Project ◽  
Probe Design ◽  
Tissue Preparation ◽  
Padlock Probe ◽  
Library Preparation ◽  
Key Steps

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

Targeted Expansion Sequencing Protocols v2

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Fei Chen ◽  
Asmamaw T. Wassie ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Pilot Project ◽  
Probe Design ◽  
Tissue Preparation ◽  
Padlock Probe ◽  
Library Preparation ◽  
Key Steps

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

Targeted ExSeq -- Tissue Preparation v2

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Asmamaw T. Wassie ◽  
Fei Chen ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Tissue Type ◽  
Model Organisms ◽  
Tissue Preparation ◽  
Enzymatic Reactions ◽  
Fixed Tissue ◽  
Tissue Sections ◽  
Antibody Staining

This protocol accompanies Expansion Sequencing (ExSeq), and describes the tissue preparation for Targeted ExSeq. The steps described here are a generalization of the protocols used for figures 4-6 of the paper, and represent our recommendations for future users of the technology. Fig. 1 shows the structure of the protocol schematically. There are three possible tissue preparation routes described in this protocol that are applicable to different experimental systems. Option (A): harvesting tissue from model organisms that can be transcardially perfused with PFA, followed by sectioning using a vibratome. We typically use this workflow for work on mouse brain sections (see figures 4-5 of ExSeq paper). Option (B): transcardially perfusing with PFA, followed by cryoprotection and cryosectioning. We occasionally use this protocol for work on mouse brain sections. Option (C): snap-freezing fresh tissue (i.e., human tumor biopsy samples, or freshly harvested tissue from mice), followed by cryoprotection and cryosectioning (see figures 2 and 6 of ExSeq paper). The final result of options (A), (B), and (C) is the preparation of fixed tissue sections (either on a glass slide or free-floating). The protocols then briefly converge for optional antibody staining, treatment with LabelX, a chemical that enables anchoring of RNA to the expansion microscopy (ExM) hydrogel, followed by casting of the the ExM gel. There are minor differences in these steps between free-floating and slide-mounted tissue sections, which are noted in the individual steps. The next step, digestion, is tissue-type dependent and may require some optimization for your tissue type. We provide two potential options here: (1) a gentle digestion for tissues such as mouse brain, and (2) a harsh digestion for non-brain tissues such as tumor biopies. The protocols then converge again for the rest of the process. After digestion, the gels are expanded and re-embedded within a second non-expanding hydrogel to lock in the sample size. The carboxylates within the expansion gel are then chemically passivated, enabling enzymatic reactions to be performed within the gel. The samples are now ready for library preparation. In more detail: Steps 1-4 describe the preparation of reagents for downstream steps. The protocol begins either along options (A)/(B), the Transcardial PFA perfusion path (Step 5, continuing to vibratome sectioning in Steps 6-7 for option (A), or cryotome sectioning in Steps 9-10 for option (B)), or along option (C), the Fresh Frozen path (Step 8, continuing to cryotome sectioning in Steps 9-10). The protocols then converge for optional antibody staining (Step 11), followed by LabelX anchoring (Step 12), optional sample trimming (Step 13), and formation of the expansion microscopy gel (Step 14). The details of the digestion step are tissue-type dependent (Step 15). The protocol then concludes with expansion (Step 16), re-embedding (Step 17), passivation, and optional trimming (Steps 18-19). This protocol was used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP). The tissue for this work was collected (see HTAPP-specific tissue collection protocol). The tissue sections were then frozen, cryosectioned, post-fixed, and permeabilized (following steps 9-10). No antibody staining was performed (skipping optional step 11). The sections were then treated with LabelX and gelled (steps 12-14). The gels were then digested using the robust digestion option in steps 15-16. The samples were then re-embedded, passivated, and trimmed (following steps 17-19).

scholarly journals Comparison of the effect of rhodium citrate-associated iron oxide nanoparticles on metastatic and non-metastatic breast cancer cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Natalia Lemos Chaves ◽  
Danilo Aquino Amorim ◽  
Cláudio Afonso Pinho Lopes ◽  
Irina Estrela-Lopis ◽  
Julia Böttner ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Breast Cancer Cells ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Cytotoxic Action ◽  
Dependent Manner ◽  
Metastatic Cells ◽  
Mcf 7

Abstract Background Nanocarriers have the potential to improve the therapeutic index of currently available drugs by increasing drug efficacy, lowering drug toxicity and achieving steady-state therapeutic levels of drugs over an extended period. The association of maghemite nanoparticles (NPs) with rhodium citrate (forming the complex hereafter referred to as MRC) has the potential to increase the specificity of the cytotoxic action of the latter compound, since this nanocomposite can be guided or transported to a target by the use of an external magnetic field. However, the behavior of these nanoparticles for an extended time of exposure to breast cancer cells has not yet been explored, and nor has MRC cytotoxicity comparison in different cell lines been performed until now. In this work, the effects of MRC NPs on these cells were analyzed for up to 72 h of exposure, and we focused on comparing NPs’ therapeutic effectiveness in different cell lines to elect the most responsive model, while elucidating the underlying action mechanism. Results MRC complexes exhibited broad cytotoxicity on human tumor cells, mainly in the first 24 h. However, while MRC induced cytotoxicity in MDA-MB-231 in a time-dependent manner, progressively decreasing the required dose for significant reduction in cell viability at 48 and 72 h, MCF-7 appears to recover its viability after 48 h of exposure. The recovery of MCF-7 is possibly explained by a resistance mechanism mediated by PGP (P-glycoprotein) proteins, which increase in these cells after MRC treatment. Remaining viable tumor metastatic cells had the migration capacity reduced after treatment with MRC (24 h). Moreover, MRC treatment induced S phase arrest of the cell cycle. Conclusion MRC act at the nucleus, inhibiting DNA synthesis and proliferation and inducing cell death. These effects were verified in both tumor lines, but MDA-MB-231 cells seem to be more responsive to the effects of NPs. In addition, NPs may also disrupt the metastatic activity of remaining cells, by reducing their migratory capacity. Our results suggest that MRC nanoparticles are a promising nanomaterial that can provide a convenient route for tumor targeting and treatment, mainly in metastatic cells.

American Society of Clinical Oncology/College of American Pathologists 2018 Focused Update of Breast Cancer HER2 FISH Testing GuidelinesResults From a National Reference Laboratory

2019 ◽  
Vol 152 (4) ◽  
pp. 479-485 ◽  
Author(s):  
Leo Lin ◽  
Deepika Sirohi ◽  
Joshua F Coleman ◽  
H Evin Gulbahce
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast ◽  
Reference Laboratory ◽  
National Reference Laboratory ◽  
Breast Cancers ◽  
National Reference ◽  
American Society ◽  
New Guidelines ◽  
Her2 Positivity

Abstract Objectives To review impact of the ASCO/CAP 2018 update on HER2 testing. Methods HER2 fluorescence in situ hybridization (FISH) test requests from primary and metastatic breast cancers between August 2018 and January 2019 were included. FISH results requiring a changed algorithm under the new guidelines (groups 2, 3, and 4) were identified and HER2:CEN17 ratios, average HER2, CEN17 signals/cell, and HER2 immunohistochemistry (IHC) results were recorded. Results Of the HER2 FISH cases 176/812(21.7%) fell within groups 2, 3, or 4; 0/12, 1/12, and 2/152 cases were positive (3+) by IHC, and 1/12, 2/12, and 6/152 cases were positive after targeted scoring from groups 2, 3, and 4, respectively. Following 2018 updates, 8.3%, 25%, and 5.3% of the groups 2, 3, and 4 were positive, respectively. Conclusions Groups 2, 3, and 4 constituted over 20% of HER2 FISH tests in a reference laboratory. The 2018 ASCO/CAP update significantly decreased the HER2 positivity rate.

In Situ Placement of High-Dose rhBMP-2 within Spine Tumors Slows Tumor Growth and Decreases Onset to Paralysis in a Rat Model of Metastatic Breast Cancer

2011 ◽  
Vol 11 (10) ◽  
pp. S3-S4
Author(s):  
Camilo Molina ◽  
Rachel Sarabia-Estrada ◽  
Ziya Gokaslan ◽  
Jean-Paul Wolinsky ◽  
Ali Bydon ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Tumor Growth ◽  
Rat Model ◽  
Metastatic Breast ◽  
High Dose ◽  
Spine Tumors

scholarly journals DCIS genomic signatures define biology and correlate with clinical outcome: a Human Tumor Atlas Network (HTAN) analysis of TBCRC 038 and RAHBT cohorts

2021 ◽  
Author(s):  
Siri H Strand ◽  
Belen Rivero-Gutierrez ◽  
Kathleen E Houlahan ◽  
Jose A Seoane ◽  
Lorraine King ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Clinical Outcome ◽  
Immune Cell ◽  
De Novo ◽  
Ductal Carcinoma ◽  
Early Stage ◽  
Expression Patterns ◽  
Human Tumor ◽  
Multiscale Approach

Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We made observations on DNA, RNA, and protein expression, and generated a de novo clustering scheme for DCIS that represents a fundamental transcriptomic organization at this early stage of breast neoplasia. Distinct stromal expression patterns and immune cell compositions were identified. We found RNA expression patterns that correlate with later events. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

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