Additional file 4 of Spatial heterogeneity of tumor microenvironment influences the prognosis of clear cell renal cell carcinoma

Additional file 4: Figure S4. (A) Heatmap showing numbers of CNs across different ccRCC tissues. Scale bar represents the number of CNs, with a maximum limit of 10. (B) Comparison of the proportions of different cell components in CNs between the scattered-CN-cold and scattered-CN-hot phenotypes. (C) Representative IMC images showing the scattered-CN-cold ccRCC tissues (P15 and P14) with the characteristic CNs

Additional file 3 of Spatial heterogeneity of tumor microenvironment influences the prognosis of clear cell renal cell carcinoma

Additional file 3: Figure S3. (A) Survival analysis between the scattered and clustered groups. (B) Comparison of clinical characteristics between the TLS-like and macrophage/T-clustered phenotypes. (C) Comparison of clinical characteristics between the scattered-CN-hot and scattered-CN-hot phenotypes. (D) Survival analysis between scattered-CN-cold, scattered-CN-hot and macrophage/T-clustered phenotypes

Additional file 1 of Spatial heterogeneity of tumor microenvironment influences the prognosis of clear cell renal cell carcinoma

Additional file 1: Figure S1. (A) Combined tSNE plot illustrating the origin of the 27 cell clusters, which are colored according to tissues. The letter A reflects the paracancerous tissue, the letter T reflects the cancerous tissue. (B) Proportions of the 27 cell clusters in total cells from the 13 ccRCC and paired paracancerous tissues. (C) tSNE plots depicting the expression of 17 markers across the 27 cell clusters, respectively. (D) Comparison of the proportion of 27 cell clusters between the ccRCC and paired paracancerous tissues

Decrease of 5-Hydroxymethylcytosine Is Associated with Progression of Hepatocellular Carcinoma through Downregulation of TET1

<div><p>DNA methylation is an important epigenetic modification and is frequently altered in cancer. Convert of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC) by ten-eleven translocation (TET) family enzymes plays important biological functions in embryonic stem cells, development, aging and disease. Recent reports showed that level of 5 hmC was altered in various types of cancers. However, the change of 5 hmC level in hepatocellular carcinoma (HCC) and association with clinical outcome were not well defined. Here, we reported that level of 5 hmC was decreased in HCC tissues, as compared with non-tumor tissues. Clincopathological analysis showed the decreased level of 5 hmC in HCC was associated with tumor size, AFP level and poor overall survival. We also found that the decreased level of 5 hmC in non-tumor tissues was associated with tumor recurrence in the first year after surgical resection. In an animal model with carcinogen DEN-induced HCC, we found that the level of 5 hmC was gradually decreased in the livers during the period of induction. There was further reduction of 5 hmC in tumor tissues when tumors were developed. In contrast, level of 5 mC was increased in HCC tissues and the increased 5 mC level was associated with capsular invasion, vascular thrombosis, tumor recurrence and overall survival. Furthermore, our data showed that expression of TET1, but not TET2 and TET3, was downregulated in HCC. Taken together, our data indicated 5 hmC may be served as a prognostic marker for HCC and the decreased expression of TET1 is likely one of the mechanisms underlying 5 hmC loss in HCC.</p></div

Additional file 6 of Spatial heterogeneity of tumor microenvironment influences the prognosis of clear cell renal cell carcinoma

Additional file 6: Table S1. Information of ccRCC tissue microarray. Table S2. Patient information for samples enrolled in the study (ccRCC tissue microarray). Table S3. Antibody panel used for acquiring images with IMC. Table S4. Details of IMC acquired images. Table S5. Cell type annotation rules. Table S6. Information of patients receiving targeted therapy and/or immunotherapy

Additional file 2 of Spatial heterogeneity of tumor microenvironment influences the prognosis of clear cell renal cell carcinoma

Additional file 2: Figure S2. (A) Heatmap representing the abundance of 7 major cells across different ccRCC tissues. The proportions were normalized by z-score for visualization. (B) Mean density curves showing the expression of 17 markers across total cells from 75 ccRCC tissues, respectively. The knee points are set as threshholds to determine the positive cells. (C) Comparison of survivals between high and low immune cell groups. High group, tissues with proportion of the immune cells ≥the third quartile (Q3); Low group, tissues with proportion of the immune cells ≤the first quartile (Q1). (D) Annotation rules for determining subtypes of T cells and macrophages

Additional file 5 of Spatial heterogeneity of tumor microenvironment influences the prognosis of clear cell renal cell carcinoma

Additional file 5: Figure S5. (A) Heatmap depicting the various immune CNs across the 5 cases with metastatic ccRCC. (B) The difference in CNs between the responder and non-responder groups. (C) Representative IMC images of non-responder (cases 4 and 5) showing the macrophage/T-clustered characteristics. (D) Representative IMC images of responders (cases 1, 2 and3) displaying the scattered-CN-hot characteristics. (E) Survival analysis between the responder and non-responder groups

Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

<div><p>Both adoptive immunotherapy and gene therapy hold a great promise for treatment of malignancies. However, these strategies exhibit limited anti-tumor activity, when they are used alone. In this study, we explore whether combination of cytokine-induced killer (CIK) adoptive immunotherapy with oncolytic adenovirus-mediated transfer of human interleukin-12 (hIL-12) gene induce the enhanced antitumor potency. Our results showed that oncolytic adenovirus carrying hIL-12 (AdCN205-IL12) could produce high levels of hIL-12 in liver cancer cells, as compared with replication-defective adenovirus expressing hIL-12 (Ad-IL12). AdCN205-IL12 could specifically induce cytotoxocity to liver cancer cells. Combination of CIK cells with AdCN205-IL12 could induce higher antitumor activity to liver cancer cells <em>in vitro</em> than that induced by either CIK or AdCN205-IL12 alone, or combination of CIK and control vector AdCN205-GFP. Furthermore, treatment of the established liver tumors with the combined therapy of CIK cells and AdCN205-IL12 resulted in tumor regression and long-term survival. High level expression of hIL-12 in tumor tissues could increase traffic of CIK cells to tumor tissues and enhance their antitumor activities. Our study provides a novel strategy for the therapy of cancer by the combination of CIK adoptive immunotherapy with oncolytic adenovirus-mediated transfer of immune stimulatory molecule hIL-12.</p> </div

Antitumor activity in the established tumor in an animal model.

<p>Tumors were established by injection of HuH-7 cells s.c. into the right flank of SCID mice. The tumor-bearing animals were treated with either 2×10⁸ ifu of different vectors by intratumoral injection or 1×10⁷ CIK by intravenous injection, or combination of both treatment. PBS was used as control group. (<b>A</b>) The size of tumor was measured every 4 days and tumor volume was calculated. The data was presented as the mean ± SEM (n = 8). The result of student <i>t</i> test shown CIK plus AdCN205-IL12 group have extremely significant difference with PBS group (<i>p</i> = 0.0062), AdCN205-GFP group (<i>p</i> = 0.0058), AdCN205-IL12 group(<i>p</i> = 0.0098), CIK group (<i>p</i> = 0.0081), and CIK plus AdCN205-GFP group (<i>p</i> = 0.0052) (<b>B</b>) Kaplan-Meier survival. The result of log-rank statistical test shown PBS group have extremely significant difference with AdCN205-GFP group (<i>p</i> = 0.009), AdCN205-IL12 group(<i>p</i> = 0.01), CIK group (<i>p</i><0.0001), CIK plus AdCN205-GFP group (<i>p</i><0.0001) and CIK plus AdCN205-Il12 group (<i>p</i><0.0001). The other group have no significant different with each other group.</p

The cytotoxcity effect of combination of CIK with oncolytic adenoviruses <i>in vitro</i>.

<p>Liver tumor cells (HuH-7 and Hep3B) were treated with CIK cells alone at effector:target ratio of 10∶1, or AdCN205-GFP, AdCN205-IL12 at the MOI of 10, or treated with both CIK at effector:target ratio of 10∶1 and AdCN205-GFP or AdCN205-IL12 at the MOI of 10. The cell survival was determined by measuring luciferase activity. Results were expressed as percentage of untreated control. The data was presented as the mean ± SD of three independent experiments.</p