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  • br Microarray Gene Expression Profiling br


    Microarray Gene Expression Profiling
    The gene expression was obtained in extreme desmoplastic CAFs (four HDEX-CAFs and four LDEX-CAFs; one HDEX-CAF went to senescence and was excluded from gene expression analysis). Early passage (passage 2) cultures of the 7 CAFs were embedded in collagen gel for 24 hours, and total RNA was isolated as described previously [21]. The gene expression microarray profiling was performed by the Princess Margaret Genomics Centre ( pmgenomics/) using the Illumina Human HT-12v4 DASL micro-array platform. The raw data from BeadChips (seven samples passed Illumina sample-dependent and -independent quality controls matrices) were preprocessed using BeadArray specific method (R package lumi v3.) for Illumina microarray [22]. The signal intensities were log2 transformed, background corrected, and normalized using robust spline normalized method. The signals were then filtered using the probe signal detection threshold (P b .05). Expression values were summarized at the gene-core/whole-transcript level based on
    HT12v4 annotation library. Preprocessed data are available through the Gene Expression Omnibus database (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE116679).
    Differentially expressed genes between HD-CAFs and LD-CAFs were identified by robust linear model analysis (fold change ≥1.5; adjusted P value b .1, R package).
    Western Blot Analysis
    The experimental procedure is described in Supplementary data.
    Stable Transfection of Polysialyltransferase (ST8SIA2) Construct into CAFs Primary Cultured Cells
    Lentiviral vector carrying ST8SIAII (pLenti-GIII-CMV-ST8SIA2) for overexpressing ST8SIA2 in HD-CAF and the control vector (pLenti-GIII-CMV-GFP) were purchased from ABM (Richmond, BC). ORF expression clone for ST8SIAII (EX-U0376-Lv122) for overexpressing ST8SIAII in LD-CAF and empty control vector (pReceiver-Lv122 (EX-NEG-lV122)) were purchased from Gene-Copoeia (Rockville, MD). Lentiviruses were prepared as described [1] by transfecting three plasmids into 293T cells: 1) pMDLg/pRRE, the vesicular stomatitis virus (VSV-G) envelope plasmid pCMV-VSG; 2) the rev-expressing plasmid pRSV-Rev; and 3) one of either of the gene transfer vector pLenti-GIII-CMV-ST8SIA2 or the empty control pLenti-GIII-CMV-GFP vector. Stocks were stored frozen at −80°C. Early passage cultures of HDEX-CAFs were infected with the ST8SIA2 lentivirus or empty control lentivirus.
    Stable knockdown of ST8SIAII gene expression in HD-CAF was established using lentiviral- shRNA vector (plentiLox; PLL 3.7. ST8SIA2 shRNA). As control, we used lentiviral- shRNA vector expressing GFP (plentiLox; PLL 3.7.GFP shRNA). The stably transfected Paxilline were selected using 1 μg/ml puromycin.
    Statistical Analysis
    Differences in tumor growth rates of xenografts between HD-CAFs and LD-CAFs and NFs were tested using linear mixed-effects model. To stabilize the residuals, a square root transformation was applied to the tumor volume. The differences in migration, invasion, and mRNA expression between HD and LD were tested by Mann-Whitney test. The Wilcoxon signed rank test was utilized for the comparison between CAFs and NFs for their gel contraction activity. For the association between the desmoplasia and the relapse rate (RR), the time to event was calculated as the duration between surgery and either relapse or death or last-follow-up. The occurrence of relapse was the event for RR. The deaths in the absence of relapses were considered competing risk. The probability of relapse was calculated with the competing risks specific techniques (cmprsk package in R 3.2.2), i.e., the estimation of the probability of relapse was based on the cumulative incidence function, and the adjusted effect of desmoplasia on relapse was tested utilizing the Fine and Gray model. Since in the UHN cohort the desmoplasia was assessed in the tumor and not necessarily in only stroma, the analysis was weighted for the amount of stroma in the tumor tissue for each patient.
    Clinical Impact of Desmoplasia on NSCLC Prognosis
    We first investigated whether high CAF content was indeed associated with more aggressive tumors. Tumors were classified into HD or LD according to histological features, mainly the percentage of DAs in the tumor stroma. The DA was defined by high proliferation 
    of fibroblasts. The estimated DA was used as a proxy for characterizing HD or LD (Figure 1). Two pathologists assessed the degree of desmoplasia in representative tumor sections of 165 NSCLC patients (UHN cohort) who had undergone curative resection. Using their consensus scores, 61 tumors (37%) were classified as HD and 104 (63%) as LD (Figure 2, A and B; Supplementary Table S1). Desmoplasia was associated with the risk for relapse in this patient cohort. The patients with HD tumors had significantly higher RR than those with LD tumors (HR = 2.45; P value = .011) (Figure 2C). The prognostic impact of desmoplasia remained significant when the model was adjusted for age, sex, stage, and tumor histology (HR = 2.47, P = .027) (Table 1). This association was stronger among adenocarcinoma patients. The patients with HD had poor outcome in comparison to the patients with LD (HR = 4.01, P = .00064, Figure 2D). The desmoplasia characteristic remained significant when the effect was adjusted for age, sex, and stage (HR = 3.69, P = .004) (Table 1).