Relationships of ESR1 and XBP1 expression in human breast carcinoma and stromal cells isolated by laser capture microdissection compared to intact breast cancer tissue

Abstract

Results from investigations of human genomics which utilize intact tissue biopsy specimens maybe compromised due to a host of uncontrolled variables including cellular heterogeneity of a sample collected under diverse conditions, then processed and stored using different protocols. To determine the cellular origin and assess relationships of mRNA expression of two genes reported to be co-expressed in human breast carcinoma (estrogen receptor-α, ESR1 and X-box binding protein 1, XBP1), gene expression analyses were performed with intact tissue sections and compared with those of laser capture microdissection (LCM)-procured carcinoma and stromal cells from serial sections of the same tissue. Frozen sections of human breast carcinomas were first evaluated for structural integrity and pathology after hematoxylin and eosin (H&E) staining. Total RNA preparations from intact tissue sections and LCM-procured carcinoma and stromal cells were reverse transcribed for measurements of ESR1 and XBP1 expression by quantitative PCR (qPCR). These results were compared with those obtained from microarray analyses of LCM-procured carcinoma cells. Levels of ESR1 and XBP1 were detected in the intact breast cancer tissue sections suggesting coordinate gene expression. Although coordinate expression of these genes was observed in the LCM-procured carcinoma cells, it was not discerned in LCM-procured stromal cells. The origin of coordinate expression of ESR1 and XBP1 observed in whole tissue sections of human breast cancer biopsies is due principally to their co-expression in carcinoma cells and not in the surrounding stromal cells as substantiated using LCM-procured cells. Collectively, a microgenomic process was established from human tissue preparation to RNA characterization and analysis to identify molecular signatures of specific cell types predicting clinical behavior.

Protein Tyrosine Phosphatase 4A2 Expression Predicts Overall and Disease-Free Survival of Human Breast Cancer and Is Associated with Estrogen and Progestin Receptor Status

“…The utilization of laser capture microdissection allowed nondestructive removal of contaminating cell types that may influence downstream analyses. This extensive data set with the associated clinical follow-up provides a unique resource for identifying specific genes or gene combinations that may alter breast cancer progression and response [16,56,57]. Few previous studies suggested that PTP4A2 expression was correlated with tumor formation, migration, and invasion in cell lines and mouse models [33,48,54] and was elevated in primary breast tumors relative to normal breast tissues [48].…”

A systems biology approach identifies a gene regulatory network in parotid acinar cell differentiation.

“…Laser capture microdissection (LCM) was performed on an Arcturus PixCell IIe LCM System (Life Technologies/ Thermofisher Scientific) [115,116]. Caps containing CapSure transfer film carrier were applied to the tissue and cells were adhered to the cap using laser pulses.…”

Microgenomic approaches to identify clinically relevant gene signatures that discriminate histologic types of breast carcinomas.

“…Since the ER and PR proteins arise from the translation of their cognate mRNA molecules, realtime reverse transcription polymerase chain reaction (RT-PCR) is a reliable technique for measuring gene expression. This assay determines the level of mRNA for the genes coding for ER (ESR1) and PR (PGR) in a sample and compares this to a control gene, producing a relative expression level (23). RT-PCR is able to specifically look for the gene coding for ERα or ERβ as well as any other known gene.…”

“…Total RNA from LCM-procured cells was isolated using PicoPure® (Arcturus/Life Technologies) kits, which are optimized for extracting RNA from cells procured by LCM (23,34). RNA in intact tissue sections, extracted with RNeasy® (Qiagen, Valencia, CA) kits, was analyzed prior to proceeding with LCM by utilization of the BioanalyzerTM instrument and reagents (Agilent Technologies, Palo Alto, CA), which estimates integrity of total RNA through analysis of the 18S and 28S rRNA profiles given by electrophoretic separation, and a RIN (RNA Integrity Number) which provides a numerical estimate of RNA integrity of the sample.…”

“…RNA in intact tissue sections, extracted with RNeasy® (Qiagen, Valencia, CA) kits, was analyzed prior to proceeding with LCM by utilization of the BioanalyzerTM instrument and reagents (Agilent Technologies, Palo Alto, CA), which estimates integrity of total RNA through analysis of the 18S and 28S rRNA profiles given by electrophoretic separation, and a RIN (RNA Integrity Number) which provides a numerical estimate of RNA integrity of the sample. Total RNA from either intact tissue sections or LCM-procured cells was reverse transcribed, and cDNA obtained was diluted 10-fold in 2 ng/μl polyinositol (Sigma) and used for the qPCR reactions (23,34). qPCR reactions were performed using a total volume of 10 μl, containing Power Sybr® Green PCR Master Mix (Applied Biosystems), forward/reverse primers and cDNA obtained from the reverse transcription reaction.…”