Journal: Cancer Research

Article Title: A Multipotent PROX1 + Tumor Stem/Progenitor Cell Population Emerges during Intestinal Tumorigenesis and Mediates Radioresistance in Colorectal Cancer

doi: 10.1158/0008-5472.CAN-23-1851

Figure Lengend Snippet: PROX1 + cells are selected upon irradiation. A–C, Uniform Manifold Approximation and Projection (UMAP; A ) of colorectal cancer organoids from patient 1, with (IR) and without (Ctrl) 2 Gy irradiation, analyzed by scRNA-seq 1 day after irradiation. B, PROX1 expression shown in a feature plot. PROX1 + cells are indicated in red; their percentages are also indicated; color intensity marks the PROX1 transcript expression level. C, A violin plot showing the PROX1 expression level in the organoids. D, Organoids isolated from tumors of colorectal cancer patients 2, 3, and 4 were irradiated with a 5-Gy daily dose on 5 consecutive days. RNA was isolated 1 day after the last irradiation. The columns show the PROX1 versus HPRT1 RNA ratio, quantified by qPCR (Ctrl = 1). Each dot represents a biological replicate, consisting of three technical replicates. E and F, A similar irradiation protocol as in D was used for SW1222 colorectal cancer cells. E, PROX1 RNA 1 day after the last irradiation dose. Each dot represents one biological replicate, which consists of two to three technical replicates. F, Proportion of PROX1 + SW1222 cells 1 day after each consecutive irradiation dose determined from a cell aliquot stained for PROX1. Seven to eight biological replicates per sample and time point were analyzed. G–J, LS174T colorectal cancer cells of volume 1 × 10 6 were injected subcutaneously into NOD/SCID gamma mice (NSG) mice (two tumors per mouse) and irradiated using either of the two irradiation protocols indicated ( G and H ). I, Quantification of the percentage of PROX1 + cells in control and irradiated tumors. Each dot indicates a single tumor. The data were pooled from two independent experiments. J, Representative images of LS174T xenografts stained for PROX1 (red) and DNA (DAPI). Scale bar, 50 µm. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Cell lines were not authenticated except for LS174T cells, which were originally authenticated by the ATCC.

Techniques: Irradiation, Expressing, Isolation, Staining, Injection, Control

Journal: Cancer Research

Article Title: A Multipotent PROX1 + Tumor Stem/Progenitor Cell Population Emerges during Intestinal Tumorigenesis and Mediates Radioresistance in Colorectal Cancer

doi: 10.1158/0008-5472.CAN-23-1851

Figure Lengend Snippet: Inhibition of LIG4 decreases the survival of PROX1 + cells following irradiation. A–D, Organoids isolated from Apc Min/+ mice or from the constitutively deleted VApc Min/+ P∆/∆ mice were plated and irradiated with a single dose of 2 to 10 Gy. A, Schematic of the experimental setup. B, Quantification of the proportion of dead (nontransparent) organoids in nonirradiated and irradiated samples. The experiment was repeated three times, each with 3 to 4 technical replicates. C and D, Representative brightfield and green fluorescent (caspase-3/7) images of the indicated treatment groups. Red and blue circles surround some of the organoids that were considered dead and alive, respectively ( D ). Scale bar, 1,000 μm. E and F, Percentage of cells expressing the indicated DDR-related transcripts in scRNA-seq data from Prox1 -positive ( Prox1 transcript >0.1; red) and Prox1 -negative (Prox1 = 0; gray) cells in Apc Min/+ tumors ( E ) and human colorectal cancer (hCRC; F ) samples. Number of Prox1 + / Prox1 − cells: Apc Min/+ , 2691/10069; human colorectal cancer, 441/5496. In the Apc Min/+ data, there are eight dots, each representing distinct scRNA-seq experiments (details in legend). The human colorectal cancer data have been pooled from 15 patients (individual patient-specific dots are not shown, GSE200997 ). G, qPCR of ID1 and ID3 in shSCR transduced (gray) and in PROX1 -silenced LS174T cells (light red), and in HCT116 cells transfected with control (black) or PROX1 -overexpressing (red) lentiviruses. The experiment was repeated three times, each with three technical replicates; two PROX1 expression vectors were used to decrease the likelihood of off-target effects. H, The percentage of PROX1 + cells in LS174T cultures treated with 10 µmol/L of SCR7 pyrazine or DMSO starting 1 day before 2 Gy irradiation and analyzed 4 day after irradiation. Growth medium supplemented with SCR7 pyrazine was replenished every 3 days. Data were pooled from three individual experiments. Each dot represents a biological replicate. I, Representative images of PROX1-stained LS174T sections from xenografts of NOD/SCID gamma mice (NSG) mice treated with SCR7 pyrazine (20 mg/kg) or PBS every day, starting 1 day before 2 Gy irradiation. The mice were euthanized 2 days after irradiation. Scale bar, 50 μm. The average proportion ±SEM of PROX1 + cells in each treatment group is shown in the figures. Statistics are as follows: **, Ctrl + IR vs. Ctrl no IR, and ^, SCR7 pyrazine no IR vs. Ctrl IR and SCR7 pyrazine + IR vs. Ctrl IR; P < 0.05. Number of mice: Control no IR, 7; SCR7 pyrazine no IR, 7; Ctrl + IR, 7; and SCR7 pyrazine + IR, 8. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

Article Snippet: Cell lines were not authenticated except for LS174T cells, which were originally authenticated by the ATCC.

Techniques: Inhibition, Irradiation, Isolation, Expressing, Transfection, Control, Staining