Approaching Solid Tumor Heterogeneity on a Cellular Basis by Tissue Proteomics Using Laser Capture Microdissection and Biological Mass Spectrometry

The purpose of this study was to examine solid tumor heterogeneity on a cellular basis using tissue proteomics that relies on a functional relationship between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS). With the use of LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lymph node containing breast carcinoma metastasis. Six specimens ∼50 000 cell each (three from tumor proper and three from tumor stroma) were collected by LCM. Specimens were processed directly on LCM caps, using sonication in buffered methanol to lyse captured cells, solubilize, and digest extracted proteins. Prepared samples were analyzed by LC/MS/MS resulting in more than 500 unique protein identifications. Decoy database searching revealed a false-positive rate between 5 and 10%. Subcellular localization analysis for stromal cells revealed plasma membrane 14%, cytoplasm 39%, nucleus 11%, extracellular space 27%, and unknown 9%; and tumor cell results were 5%, 58%, 26%, 4%, and 7%, respectively. Western blot analysis confirmed specific linkage of validated proteins to underlying pathology and their potential role in solid tumor heterogeneity. With continued research and optimization of this method including analysis of additional clinical specimens, this approach may lead to an improved understanding of tumor heterogeneity, and serve as a platform for solid tumor biomarker discovery

DS_10.1369_0022155419882292 – Supplemental material for CTLA-4 Immunohistochemistry and Quantitative Image Analysis for Profiling of Human Cancers

Supplemental material, DS_10.1369_0022155419882292 for CTLA-4 Immunohistochemistry and Quantitative Image Analysis for Profiling of Human Cancers by Charles Brown, Farzad Sekhavati, Ruben Cardenes, Claudia Windmueller, Karma Dacosta, Jaime Rodriguez-Canales and Keith E. Steele in Journal of Histochemistry & Cytochemistry</p

Approaching Solid Tumor Heterogeneity on a Cellular Basis by Tissue Proteomics Using Laser Capture Microdissection and Biological Mass Spectrometry

The purpose of this study was to examine solid tumor heterogeneity on a cellular basis using tissue proteomics that relies on a functional relationship between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS). With the use of LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lymph node containing breast carcinoma metastasis. Six specimens ∼50 000 cell each (three from tumor proper and three from tumor stroma) were collected by LCM. Specimens were processed directly on LCM caps, using sonication in buffered methanol to lyse captured cells, solubilize, and digest extracted proteins. Prepared samples were analyzed by LC/MS/MS resulting in more than 500 unique protein identifications. Decoy database searching revealed a false-positive rate between 5 and 10%. Subcellular localization analysis for stromal cells revealed plasma membrane 14%, cytoplasm 39%, nucleus 11%, extracellular space 27%, and unknown 9%; and tumor cell results were 5%, 58%, 26%, 4%, and 7%, respectively. Western blot analysis confirmed specific linkage of validated proteins to underlying pathology and their potential role in solid tumor heterogeneity. With continued research and optimization of this method including analysis of additional clinical specimens, this approach may lead to an improved understanding of tumor heterogeneity, and serve as a platform for solid tumor biomarker discovery

Analysis of Transcription Factor mRNAs in Identified Oxytocin and Vasopressin Magnocellular Neurons Isolated by Laser Capture Microdissection

<div><p>The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV) vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun) are differentially expressed in the Oxt and Avp MCNs.</p></div

Specificity of Expression of the Oxytocin and vasopressin rAAV constructs in the SON.

<p>A. The 440OTIII-EGFP rAAV that was injected into the SON contains 440 bp of the promoter sequence upstream of the transcription start site (TSS) of the oxytocin gene, all three exons, fused to the EGFP sequence within the 3rd exon (EX 3), followed by 768 bp of the 3′UTR of the oxytocin gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069407#pone.0069407-Fields1" target="_blank">[17]</a>. The immunohistochemical (IHC) image shown below the construct shows that the EGFP from the 440 OTIII-EGFP virus is specifically expressed in Oxt MCNs in the dorsal SON. The green color represents the fluorescence of the EGFP immunoreactivity in Oxt MCNs and the red fluorescence depicts the PS 41 antibody immunoreactivity in the Avp MCNs. Scale line for both panels A and B is shown in lower panel B. B. The 2.0VPI-EGFP rAAV that was injected contains 2.0 bp of the promoter sequence found upstream of the vasopressin gene TSS, exon 1 of the vasopressin gene, fused to the EGFP sequence, and 173 bp of the 3′UTR downstream of the Avp gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069407#pone.0069407-Ponzio1" target="_blank">[18]</a>. The immunohistochemical (IHC) image shown below the construct shows that the EGFP from the 2.0VPI-EGFP virus is specifically expressed in Avp MCNs in the ventral SON. The green color represents the fluorescence of the EGFP immunoreactivity in Avp MCNs and the red fluorescence depicts the PS 38 antibody immunoreactivity in the Oxt MCNs. Abbreviation: OX, optic chiasm.</p

Supplementary Figures 1 - 4, Tables 1 - 3 from Essential Role of Aldehyde Dehydrogenase 1A3 for the Maintenance of Non–Small Cell Lung Cancer Stem Cells Is Associated with the STAT3 Pathway

PDF file - 465K, Supplementary Table S1. The correlation between ALDH1A3 protein expression and percentage of ALDH+ cells in NSCLC lines (Data for Figure 2D). Supplementary Table S2. Incidence of xenograft tumors derived from control or ALDH1A3 knockdown lung cancer cells. Supplementary Table S3. The association between ALDH1A3 protein expression and clinicopathological variables for 455 NSCLC patient samples. Figure S1. ALDH profiling of 8 NSCLC lines using the flow cytometry based Aldefluor assay. Figure S2. Anchorage dependent colony formation efficiency was determined by growing serial dilutions of sorted ALDH+ and ALDH- cells for 2 weeks in liquid culture. Figure S3. A, ALDH1A3 protein expression in a set of NSCLC lines was detected by western blots. The corresponding percentage of ALDH+ cells was analyzed by Aldefluor assay. B, western blot showed ALDH1A1 protein expression in certain HBEC and NSCLC lines. C, ALDH1A3 was knocked down using three siRNAs in a panel of NSCLC lines with different driver mutations followed by liquid colony formation assays. RT-PCR was performed to examine ALDH1A3 mRNA expression. Figure S4. Ectopic expression of ALDH1A3 does not affect tumorigenicity of lung cancer cells.</p

Supplementary Material from An Expression Signature as an Aid to the Histologic Classification of Non–Small Cell Lung Cancer

Extended HTG EdgeSeq methodology</p

View of brain sections on the Frame metal slide LCM slides, and the LCM Cap position.

<p>A. Illustration of three metal frame membrane slides each containing four hypothalamic sections mounted on the stage of the Arcturus XT instrument ready for LCM. B. Schematic view of the Metal Frame Membrane slide and LCM cap. With these slides the LCM cap sits directly on the membrane without touching the tissue. This allows the cap to sit evenly without being affected by any folds in the tissue and adjustments to the laser’s power and alignment usually do not need to be made. Use of the Macro LCM caps also reduces the danger of contaminating the sample with surrounding tissue.</p

Supplementary Table 1 from An Expression Signature as an Aid to the Histologic Classification of Non–Small Cell Lung Cancer

Gene Lists for the ADC-SCC and Tumor-Nonmalignant Signatures</p

Calculated Fold Differences of Oxt vs. Avp MCN mRNA.

<p>Calculated Fold Differences of Oxt vs. Avp MCN mRNA.</p