DataSheet_1_A nomogram based on the preoperative neutrophil-to-lymphocyte ratio to distinguish sarcomatoid renal cell carcinoma from clear cell renal cell carcinoma.docx

ObjectiveOur study aimed to assess the predictive value of the preoperative neutrophil-to-lymphocyte ratio(NLR) in distinguishing sarcomatoid renal cell carcinoma (SRCC) from clear cell renal cell carcinoma(CCRCC) and to developing a nomogram based on the preoperative NLR and other factors to distinguish SRCC from CCRCC.Materials and methodsThe database involved 280 patients, including 46 SRCC and 234 CCRCC. logistic analysis was conducted to select the variables associated with identifying SRCC preoperatively, and subgroup analysis was used to further validate the ability of NLR with preoperative identification of SRCC.In addition, The data were randomly separated into a training cohort(n=195) and a validation cohort(n=85). And an NLR-based nomogram was plotted based on the logistic analysis results. The nomogram was evaluated according to its discrimination, consistency, and clinical benefits.ResultsMultivariate analysis indicated that NLR, flank pain, tumor size, and total cholesterol(TC) were independent risk factors for identifying SRCC. The results of subgroup analysis showed that higher NLR was associated with a higher probability of SRCC in most subgroups. The area under the curve(AUC) of the training and validation cohorts were 0.801 and 0.738, respectively. The results of the calibration curve show high consistency between predicted and actual results. Decision Curve Analysis(DCA) showed clinical intervention based on the model was beneficial over most of the threshold risk range.ConclusionNLR is a potential indicator for preoperative differentiation of SRCC and CCRCC, and the predictive model constructed based on NLR has a good predictive ability. The new model could provide suggestions for the early identification of SRCC.</p

The differential inhibition of proliferation between HK-2 and 7860 cells in differing culture conditions.

<p>The differential inhibition rate is the difference between inhibition rate of proliferation of 7860 and HK-2 cells by different culture conditions. *<i>p</i><0.05.</p

Cellular proliferation under various experimental conditions.

<p>Proliferation of HK-2 and 7860 cells after: A, cold-culture (4°C) in UW solution; B, cold-culture in normal medium; and C, culture at 37°C in UW solution. Cells were plated onto 96-well plates at 3000 cells per well in complete medium and cultured for 24 h in a 37°C, 5% CO₂ incubator. The culture medium was then changed, and wells were divided equally into four groups. Groups 1, 2, 3, and 4 were cold-cultured in UW solution for 0, 6, 12, and 24 h, respectively, as indicated. Subsequently, 10 µl of CCK solution was added to each well, and plates were returned to the incubator for 4 h before the absorbance at 450 nm was measured. *<i>p</i><0.05.</p

Expression of Ki67 following organ preservation.

<p>Protein samples were separated by SDS-PAGE, transferred onto membranes, and probed with anti-Ki67 antibodies. Stripped membranes were re-probed with anti-GAPDH antibodies as a loading control.</p

Kidneys with small renal cancer after perfusion with 4°C UW solution.

<p>All three renal cancers were less than 4.8(ccCRC).</p

Proliferation of HK-2 and 7860 cells after cold-culture in UW solution and recovery in normal conditions.

<p>Cells were cold-cultured in UW solution for the indicated length of time and harvested. Harvested cells were plated onto 96-well plates at 3000 cells per well in complete medium and allowed to grow for 20 h in a 37°C, 5% CO₂ incubator. Subsequently, 10 µl of CCK solution was added to each well and plates were returned to the incubator for another 4 h before the absorbance at 450 nm was measured. *<i>p</i><0.05.</p

Percentage of viable cells after cold-storage in UW solution or normal saline.

<p>After culture for the indicated length of time, viable and dead cells were counted by Trypan Blue exclusion and microscopy. *<i>p</i><0.05.</p

DataSheet1_Sarcomatoid-associated gene risk index for clear cell renal cell carcinoma.xlsx

Sarcomatoid renal cell carcinoma is a de-differentiated form of kidney cancer with an extremely poor prognosis. Genes associated with sarcomatoid differentiation may be closely related to the prognosis of renal cell carcinoma. The prognosis of renal cell carcinoma itself is extremely variable, and a new prognostic model is needed to stratify patients and guide treatment. Data on clear cell renal cell carcinoma with or without sarcomatoid differentiation were obtained from TCGA database, and a sarcomatoid-associated gene risk index (SAGRI) and column line graphs were constructed using sarcomatoid-associated genes. The predictive power of the SAGRI and column line graphs was validated using an internal validation set and an independent validation set (E-MTAB-1980). The SAGRI was constructed using four sarcoma-like differentiation-related genes, COL7A1, LCTL, NPR3, ZFHX4, and had a 1-year AUC value of 0.725 in the training set, 0.712 in the internal validation set, and 0.770 in the independent validation set for TCGA training cohort, with high model reliability. The molecular characteristics among the SAGRI subgroups were analyzed by multiple methods, and results suggested that the SAGRI-HIGH subgroup may benefit more from immunotherapy to improve prognosis. SAGRI satisfactorily predicted the prognosis of patients with clear cell renal cell carcinoma with or without sarcomatoid differentiation.</p

Expression levels of EMT-associated proteins in PCa cells following knockdown of HDGF.

<p>The relative protein expression levels of Vimentin, N-cadherin, Snail and Slug were reduced, and E-cadherin was increased in shRNA-HDGF transfected (A) DU145, (C) PC3 and (E) LNCaP cells compared with their corresponding control cells transfected with shRNA-CTR or treated with PBS. The quantification of EMT-associated protein gray intensity of (B) DU145, (D) PC3 and (F) LNCaP cells in each group are presented. The data are presented as mean ± standard deviation of three independent experiments. The relative protein expression level was normalized to β-actin. *<i>P</i> < 0.05 vs PBS, **<i>P</i> < 0.05 vs shRNA-CTR. EMT, epithelial mesenchymal transition; PCa, prostate cancer; HDGF, hepatoma-derived growth factor; shRNA-HDGF, group transfected by recombinant lentivirus shRNA targeting HDGF sequence; shRNA-CTR, group transfected by recombinant lentivirus shRNA with scrambled sequence; PBS, group treated by PBS.</p