Supplementary Figures from Autotaxin–Lysophosphatidic Acid Signaling Axis Mediates Tumorigenesis and Development of Acquired Resistance to Sunitinib in Renal Cell Carcinoma
Authors: Su Shih-Chi , Hu Xiaoxiao , Kenney Patrick A. , Merrill Megan M. , Babaian Kara N. , Zhang Xiu-Ying , Maity Tapati , Yang Shun-Fa , Lin Xin , Wood Christopher G. 
Published: April 2023

<p>PDF file 353K, Supplemental Figure 1. Identification of ATX/ENPP2 in sunitinib-treated RCC endothelium. (A) Gene expression analyses of known endothelial markers between anti-CD31-coated magnetic beads-bound renal endothelial population and unbound renal cells. (B) A cluster with the most significant difference in endothelial gene expression between sunitinib-treated and -untreated human RCC specimens. Supplemental Figure 2. LPA induces cell proliferation in RCC, but not in HUVECs. UMRC3 and HUVECs were cultured with medium only (CTL), 20 microM LPC, or 1 microM LPA. At the indicated times, cell proliferation was measured by MTT assay. Results are shown as percent of untreated control. Supplemental Figure 3. LPA stimulation of pro-angiogenic factor expression in RCC cells. RCC cell lines (UMRC3, 786-O, and Caki-1) and primary culture (HRC-223) were serum-starved for 24 hours and treated with 1 microM LPA for 3 hours. Total RNA was prepared and real-time PCR was performed for the indicated genes. Data are expressed as mean {plus-minus} S.D. of three replicates and are representative of two separate experiments. Supplemental Figure 4. Establishment of RCC xenograft models with the acquired resistance to sunitinib. (A) Animals were injected s.c. into the flanks with UMRC3 and treated daily with water (control) and 40 mg/kg sunitinib by oral gavage. Tumor measurement was performed twice per week. Upon the treatment, tumor growth showed an initial plateau during which the tumors stopped growing and then began to actively shrink. This period of active shrinkage is referred to as the sensitive phase. Tumors that shrank initially when the treatment began and showed a long-term trend toward continued growth later, were considered sunitinib-resistant. Arrowhead indicates the time point at which sunitinib treatment began. (B) Tumors under the sensitive and resistant phase of treatment were collected and subjected to immunohistochemistry by using monoclonal antibodies against CD34. (C) The microvascular density was calculated from three independent tumor sections per group and expressed as the mean plus-minus S.D. (Student's t-test; *, p<0.05 compared with untreated controls); scale bars, 50 microm. Supplemental Figure 5. Effects of Sunitinib on sprouting angiogenesis. (A-C) HUVECs were allowed to invade into 3D collagen matrices (2.5 mg/ml) containing 0.1 microM S1P in the absence (CTL) or presence of 40 ng/ml VEGF, IL-8, or bFGF combined with 0.1 microM or 1 microM Sunitinib. After 24 hours, cultures were fixed, stained, photographed (A) and quantified for invasion density (B), distance (C). For invasion density, data represent average numbers of invading cells per standardized field (n=3 fields). For invasion distance, data from 20 cells were averaged and are presented as means {plus-minus} SE. (**P<0.01 compared with vehicle control using Student t-test; nd; not determined)</p>