Patient-Derived Colorectal Cancer Organoids Upregulate Revival Stem Cell Marker Genes following Chemotherapeutic Treatment
<p>Patient-derived cancer organoids recapitulate the histopathological characteristics of their primary tumours and inter-tumoural heterogeneity in stem cell signatures. (<b>A</b>): Haematoxylin and eosin (H&E) staining of sectioned tissue from primary colorectal adenocarcinoma and patient-derived cancer organoids derived from the same tumour. Scale bar, 400 μm. (<b>B</b>): Immunohistochemical detection of CDX2 (marker of adenocarcinomas of intestinal origin) in primary colorectal adenocarcinoma compared to patient-derived colorectal cancer organoids (PDCOs). Scale bar, 400 μm. (<b>C</b>): Immunohistochemical detection of LGR5 (CBC stem cell marker) and CK20 (intestinal epithelial marker) in the colorectal adenocarcinomas and PDCOs. Scale bar, 400 μm. (<b>D</b>): The expression levels (2<sup>−ΔCt</sup>) for <span class="html-italic">LGR5, EPHB2, BMI1, CLU, ANXA1</span> and <span class="html-italic">KRT20</span> were calculated relative to beta-2-microglobulin and β-actin by qRT-PCR in individual PDCO lines.</p> "> Figure 2
<p>Elevated expression of a subset of stem cell markers correlates with resistance to chemotherapy. (<b>A</b>): Representative brightfield images of patient-derived cancer organoids (PDCOs) in response to chemotherapeutic treatment with 5-fluorouracil (5-FU) and the immunohistochemical detection of the active cleaved form of caspase 3 (apoptotic marker) in 10 µM treated PDCOs. Scale bar, 500 μm. (<b>B</b>): Dose-response curve of PDCOs in response to chemotherapeutic treatment with 5-FU (<span class="html-italic">n</span> = 6, mean ± SEM). (<b>C</b>): Area under the curve (AUC) analysis of 5-FU sensitivity in six patient-derived tumoroids. (<b>D</b>): Linear regression correlation analysis between the expression of stem cell marker genes and AUC showing the best-fit line (solid line) and 95% prediction interval (dash-line). (<b>E</b>): Expression of stem cell marker genes in PDCOs in response to an increasing dose of 5-FU. Fold change is calculated by the average gene expression levels (2<sup>−ΔCt</sup>) relative to the vehicle control (<span class="html-italic">n</span> = 6, mean ± SEM). In each graph, asterisks indicate pairs of means (compared to vehicle control) that were significantly different using Mann–Whitney test (*, <span class="html-italic">p</span> < 0.05; **, <span class="html-italic">p</span> < 0.01; ***, <span class="html-italic">p</span> < 0.001).</p> "> Figure 3
<p><span class="html-italic">CLU</span> expression in colon adenocarcinoma patients is associated with decreased overall survival. (<b>A</b>): Kaplan–Meier survival plot comparing The Cancer Genome Atlas (TCGA) colon adenocarcinoma patients with high (<span class="html-italic">n</span> = 110) and low (<span class="html-italic">n</span> = 110) expression of <span class="html-italic">CLU</span> using the OncoLnc tool. The associated log-rank <span class="html-italic">p</span>-value is 0.0286. (<b>B</b>): Kaplan–Meier survival plot for high- (red, <span class="html-italic">n</span> = 121) and low- (green, <span class="html-italic">n</span> = 122) risk groups in GSE41258 database by SurvExpress tool shows cumulative survival against time (months) and the box plot shows the corresponding <span class="html-italic">CLU</span> expression across groups. The number of individuals, the number of censored, and the CI of each risk group are shown in the top-right insets. Censoring samples are shown as “+” marks. (<b>C</b>): Kaplan–Meier survival plot for high- (red, <span class="html-italic">n</span> = 95) and low- (green, <span class="html-italic">n</span> = 94) risk groups in GSE41258 database by SurvExpress tool shows cumulative recurrence-free survival against time (months) and the box plot shows the corresponding <span class="html-italic">CLU</span> expression across groups. The number of individuals, the number of censored, and the CI of each risk group are shown in the top-right insets. Censoring samples are shown as “+” marks.</p> ">
Abstract
:1. Introduction
2. Experimental Section
2.1. Ethics and Consent
2.2. Patient Data
2.3. Establishing Colorectal Cancer Organoids
2.4. Organoid Drug Sensitivity Testing
2.5. Histological Sections
2.6. Immunohistochemistry
2.7. Quantitative RT–PCR Analysis
2.8. Survival Analysis
3. Results
3.1. Patient-Derived Colorectal Cancer Organoids Recapitulate the Histopathological Characteristics of Their Primary Tumours and Display Inter-Tumoural Heterogeneity in Stem Cell Signatures
3.2. Elevated Expression of a Subset of Stem Cell Markers Correlates with Resistance to Chemotherapy
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Antibodies/Dye | Host | Dilution | Supplier | Cat/Lot number |
---|---|---|---|---|
Primary | ||||
Cleaved Caspase 3 (Asp175) | Rabbit | 1:250 | Cell Signaling | 9661S |
Cytokeratin 20 (CK20) (SP33) | Rabbit | 1:1 | Roche Ventana | 790-4431 |
Caudal type homeobox 2 (CDX2) | Rabbit | 1:1000 | Abcam | Ab76541 |
Leucine rich repeat containing G protein-coupled receptor 5 (LGR5) | Rabbit | 1:200 | Genentech | n/a |
Secondary | ||||
Anti-rabbit horseradish peroxidase | Goat | 1:200 | Life Technologies | G21234 |
Gene | Forward Primer Sequences | Reverse Primer Sequences | Product Length (bp) |
---|---|---|---|
ACTB | CTGGCACCACACCTTCTACAATG | GGTCTCAAACATGATCTGGGTC | 124 |
ANXA1 | TTTGCAAGAAGGTAGAGATAAAGAC | GGATGACTTCACAGTTTGAACAT | 121 |
B2M | GTGCTCGCGCTACTCTCTC | GTCAACTTCAATGTCGGAT | 142 |
BMI1 | GGTACTTCATTGATGCCACAACC | CTGGTCTTGTGAACTTGGACATC | 104 |
CLU | CAGGCCATGGACATCCACTT | GTCATCGTCGCCTTCTCGTA | 78 |
EPHB2 | TTGGGCTCTCACGCTTTCTA | AGGTGAACTTCCGGTACTGG | 120 |
Ki67 | CAGCACCTGCTTGTTTGGAAG | TAATATTGCCTCCTGCTCATGGAT | 109 |
KRT20 | CTGAGGTTCAACTAACGGAGCTG | AACAGCGACTGGAGGTTGGCTA | 129 |
LGR5 | CCTTCCAACCTCAGCGTCTT | AGGGATTGAAGGCTTCGCAA | 250 |
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Engel, R.M.; Chan, W.H.; Nickless, D.; Hlavca, S.; Richards, E.; Kerr, G.; Oliva, K.; McMurrick, P.J.; Jardé, T.; Abud, H.E. Patient-Derived Colorectal Cancer Organoids Upregulate Revival Stem Cell Marker Genes following Chemotherapeutic Treatment. J. Clin. Med. 2020, 9, 128. https://doi.org/10.3390/jcm9010128
Engel RM, Chan WH, Nickless D, Hlavca S, Richards E, Kerr G, Oliva K, McMurrick PJ, Jardé T, Abud HE. Patient-Derived Colorectal Cancer Organoids Upregulate Revival Stem Cell Marker Genes following Chemotherapeutic Treatment. Journal of Clinical Medicine. 2020; 9(1):128. https://doi.org/10.3390/jcm9010128
Chicago/Turabian StyleEngel, Rebekah M., Wing Hei Chan, David Nickless, Sara Hlavca, Elizabeth Richards, Genevieve Kerr, Karen Oliva, Paul J. McMurrick, Thierry Jardé, and Helen E. Abud. 2020. "Patient-Derived Colorectal Cancer Organoids Upregulate Revival Stem Cell Marker Genes following Chemotherapeutic Treatment" Journal of Clinical Medicine 9, no. 1: 128. https://doi.org/10.3390/jcm9010128