Is BMI-1 important for tumor growth based off of the results that are exhibited

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Is BMI-1 important for tumor growth based off of the results that are exhibited in figure 1d? Please explain.

BMI-1 knockdown reduces human colorectal cancer growth We detected BMI-1 expression in most cells of a well-characterized human colon adenocarcinoma cell line, LS174T, and five patientderived colorectal tumor samples under conditions that permitted expansion in vitro (Fig. 1a,b). When we sorted these samples into phenotypic fractions enriched for colorectal CICs and non-CICs, BMI-1 was expressed in both fractions, similar to other markers used to isolate normal intestinal stem cells and colorectal CICs (Supplementary Figs. 1–3). To determine whether reduction of BMI-1 levels affected the growth of human colorectal cancer cells, we stably knocked down BMI-1 transcript levels using a total of four different lentivirus encoded shRNAs. The different shRNAs yielded similar results, and we employed one shRNA for most studies, as it reliably yielded cells with >60% BMI-1 knockdown at the mRNA and protein levels (BMI-1-KD cells) (Fig. 1a,b). In addition to an shRNA to BMI1 (shBMI-1), the lentivirus also encoded eGFP. BMI-1-KD cells were progressively outcompeted and lost their ability to grow in vitro, as evidenced by a decline in the fraction of shBMI-1–expressing eGFP+ cells over time in both cell lines and primary patient-derived cell cultures (Fig. 1c). To evaluate effects on in vivo tumor-forming potential, we injected BMI-1-KD cells subcutaneously into nonobese diabetic– severe combined immunodeficiency (NOD-SCID) and interleukin-2 receptor ?c–deficient (NSG) mice. Four weeks following transplantation, BMI-1-KD LS174T cells displayed a 12-fold reduction in tumor growth as compared to control cells expressing an shRNA to luciferase (shLUC) (P < 0.0001, Fig. 1d). Likewise, the mass of tumors from patient-derived colorectal cell cultures was significantly reduced in the BMI-1-KD group of sample 02 (P < 0.0001, Fig. 1d) and sample 04 (P = 0.02, Fig. 1d); no tumors were detected in mice given BMI-1-KD sample 01 cells (Fig. 1d). We obtained similar results using additional shRNAs targeting BMI-1 (Supplementary Fig. 4a). To ensure that the dependence on BMI-1 for tumor cell growth was not due to short-term culturing, we generated single-cell suspensions from five additional colorectal cancer specimens immediately following surgical resection and directly transduced them without expansion in vitro. We observed considerably reduced tumor growth following injection under the renal capsule in the BMI-1-KD group compared to the control transduced cells expressing shLUC for three of the five tested samples (Fig. 1e). For sample 05 and LS174T cells, BMI-1-KD cells generated smaller tumors upon re-injection into secondary recipients, indicating that BMI-1-KD cells exhibit a sustained reduction in tumor growth (Fig. 1e and Supplementary Fig. 4b). Collectively, these data indicate that human colorectal cancer cells require BMI-1 to generate and maintain tumor growth in vivo. The observed decrease in tumor mass

Figure 1 BMI-1 knockdown impairs human colorectal cancer cell growth. (a) BMI-1 transcript levels in LS174T cells transduced with the shBMI-1 knockdown vector (normalized to GAPDH). (b) Immunoblots showing BMI-1 and GAPDH protein in lysates of LS174T colon adenocarcinoma cell line and five patient colorectal tumor samples transduced with the shLUC or shBMI-1 vectors. (c) The percentage of cells expressing eGFP among human colorectal cancer cells transduced with the shBMI-1 or shLUC virus and passaged every 4–7 d. For LS174T cells, data are expressed as mean ± s.e.m. of triplicate cultures. (d) eGFP+ tumor weight of control and BMI-1-KD colorectal cancer cells that were grown subcutaneously in immunocompromised mice. Tumor weight was calculated by multiplying tumor mass by percentage eGFP expression, which was similar between shLUC and shBMI-1 cells before injection. Each circle represents one mouse, and horizontal lines indicate the mean tumor weight. (e) eGFP+ tumor growth in immunodeficient mice injected in the renal capsule with primary human colorectal cancer cells that were not expanded in vitro and that were transduced with control or shBMI-1 lentiviruses. Circles represent the weight of one tumor, and horizontal lines show the mean (*P < 0.05, **P < 0.01 and ***P < 0.001).

C LS174T shLUC LS174T shBMI-1 1.0 0.8 ? 0.6 0.4 0.2 100 80 60 40 20 ?Sample 01 shLUC Sample 01 shBMI-1 Sample 02 shLUC Sample 02 shBMI-1 Sample 03 shLUC Sample 03 shBMI-1 100 BMI-1 60 1 GAPDH 20 LS174T Sample 02 Sample 10 Sample 03 Sample 11 Sample 01 , ' 100% 89% 99% 91% 74% 64% % KD 0 10 20 30 40 50 60 Time in vitro (d) 0 20 40 60 80 100 Time in vitro (d) Sample 01 Sample 04 e Sample 05 Sample 06 Sample 07 LS174T Sample 02 500 400- oo 300 200 100-s 5,000 4,000 3,000 2,000 1,000- 2,000 1,500 1,000 4,500 500 12,000 10,000 o 800 4,000 2,000 ooo 1,000 E 400o 600-o 8,000 6,000 4,000 o 2,000 o 300 S 400 ke 200o o 500o 100- No 0

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Is BMI-1 important for tumor growth based off of the results that are exhibited

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