Patient-Derived Colorectal Cancer Organoids Upregulate Revival Stem Cell Marker Genes following Chemotherapeutic Treatment

Abstract

Colorectal cancer stem cells have been proposed to drive disease progression, tumour recurrence and chemoresistance. However, studies ablating leucine rich repeat containing G protein-coupled receptor 5 (LGR5)-positive stem cells have shown that they are rapidly replenished in primary tumours. Following injury in normal tissue, LGR5+ stem cells are replaced by a newly defined, transient population of revival stem cells. We investigated whether markers of the revival stem cell population are present in colorectal tumours and how this signature relates to chemoresistance. We examined the expression of different stem cell markers in a cohort of patient-derived colorectal cancer organoids and correlated expression with sensitivity to 5-fluorouracil (5-FU) treatment. Our findings revealed that there was inter-tumour variability in the expression of stem cell markers. Clusterin (CLU), a marker of the revival stem cell population, was significantly enriched following 5-FU treatment and expression correlated with the level of drug resistance. Patient outcome data revealed that CLU expression is associated with both lower patient survival and an increase in disease recurrence. This suggests that CLU is a marker of drug resistance and may identify cells that drive colorectal cancer progression.

Keywords: bowel cancer; chemotherapy resistance; colon; colorectal; organoid; stem cell; tumoroid.

Figure 1

Patient-derived cancer organoids recapitulate the histopathological characteristics of their primary tumours and inter-tumoural heterogeneity in stem cell signatures. (A): 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): 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. (C): Immunohistochemical detection of LGR5 (CBC stem cell marker) and CK20 (intestinal epithelial marker) in the colorectal adenocarcinomas and PDCOs. Scale bar, 400 μm. (D): The expression levels (2^−ΔCt) for LGR5, EPHB2, BMI1, CLU, ANXA1 and KRT20 were calculated relative to beta-2-microglobulin and β-actin by qRT-PCR in individual PDCO lines.

Figure 2

Elevated expression of a subset of stem cell markers correlates with resistance to chemotherapy. (A): 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): Dose-response curve of PDCOs in response to chemotherapeutic treatment with 5-FU (n = 6, mean ± SEM). (C): Area under the curve (AUC) analysis of 5-FU sensitivity in six patient-derived tumoroids. (D): 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). (E): 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^−ΔCt) relative to the vehicle control (n = 6, mean ± SEM). In each graph, asterisks indicate pairs of means (compared to vehicle control) that were significantly different using Mann–Whitney test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3

CLU expression in colon adenocarcinoma patients is associated with decreased overall survival. (A): Kaplan–Meier survival plot comparing The Cancer Genome Atlas (TCGA) colon adenocarcinoma patients with high (n = 110) and low (n = 110) expression of CLU using the OncoLnc tool. The associated log-rank p-value is 0.0286. (B): Kaplan–Meier survival plot for high- (red, n = 121) and low- (green, n = 122) risk groups in GSE41258 database by SurvExpress tool shows cumulative survival against time (months) and the box plot shows the corresponding CLU 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. (C): Kaplan–Meier survival plot for high- (red, n = 95) and low- (green, n = 94) risk groups in GSE41258 database by SurvExpress tool shows cumulative recurrence-free survival against time (months) and the box plot shows the corresponding CLU 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.