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6. To study the behaviors of metastatic cancer cells, GFP-expressing cancer cells were prepared from aggressive mammary tumors from transgenic mice. These cells were injected into the tail veins of tumor-free host mice and the proportion of GFFP+ cells relative to total cells in the lungs were followed over the indicated time intervals (shown on the right). Initially, -0.1% of all cells in the lungs were GFP+ carcinoma cells, which declined to -0.03% a week later and then increased progressively to ~1% by week 4. 3 hr 1 week 2 weeks 4 weeks Which of the following is TRUE about the results? a. The ~1% GFP+ cells in the lungs 3 hours after injection was due to the fast colonization of the cancer cells in the lung. b. The drop of GFP+ cells in the lung at week 1 was due to immune rejection. C. The increase of GFP+ cells from week 1 to week 2 was due to cancer colonization. d. The increase of GFP+ cells from week 2 to week 4 was due to the transformation of lung cells in the host by the injected cancer cells. % GFP+ cells/total cells in lungs

(A) Twist EMT-TF 104 100 104 SCS 8. A group of transcription factors, including Snail, Slut, and Twist, are found to express in the embryonic cells that undergo EMT. Thus, they are called EMT-TF. Figure on the right are data from an experiment to test the role of EMT-TF in differentiated epithelial cells. HMLE HMLE+ HMLE+ Snail EMT-TF When cultured immortalized human mammary epithelial cells (HMLE) were forced to express high level of EMT-TF, either 0 Snail (the HMLE+Snail EMT-TF group) or Twist (the HMLE+Twist EMT-TF group), the cells changed to fibroblast shape (top 101 102 103 104 100 101 102 103 104 100 101 102 103 104 pictures). When these cells were subjected to low high flow cytometry, analysis, more CD44+;CD24- cells were found (the upper left populations in the flow sytometry.graphs) 103 10? 107 102 102 102 CD44 101 10' 10 non SCS 100 100 CD24 a. Which of the following is NOT a correct interpretation for these results? EMT-TF can turn differentiated cells back to stem cells. b. EMT-TF can turn immortalized cells into cancer cells. EMT-TF can turn epithelial cells into mobile mesenchymal cells. d. EMT-TF can enhance tumor metastasis. c.

165 160 140 117 106 96 92 85 9. To study the role of EMT-TF in tumor metastasis, the highly metastatic 4T1 mouse mammary carcinoma cells were implanted into the mammary gland of normal mice and later the number of metastatic nodules in the lung was counted. The results are shown on the right. In the experimental group, mice were implanted with 4T1 cells that had been infected with an engineered retrovirus expressing siRNA specific against the Twist EMT-TF to knockdown the expression of Twist (4T1 Twist-siRNA). Mice in the control group received 4T1 cells infected with control siRNA. Another control is mice receiving non- engineered 4T1 cells (the far right bar on the graph). number of metastatic nodules per lung 120 100 80 60 40 54 20 18 18 20 16 JY1192 JY1193 JY1194 JY1195 JY1196 JY1197B JY1198 JY1200 JY1072 JY1073 JY1074 JY1075 A336 A337 4T1 4T1 control-siRNA 105 4T1 Twist-siRNA average no. 14 of metastatic nodules per lung a. Which of the following is a CORRECT interpretation for the results? Twist is required for mammary carcinoma metastasis. b. Twist siRNA selectively caused cell death in the carcinoma stem cells. Twist siRNA somehow caused the mammary carcinoma to metastasize to other organs. d. Knockdown of Twist caused carcinoma cells to grow smaller and not get trapped in the lung capillaries. C.