Table_1_Case Report: Durable complete response of metastatic hepatocellular carcinoma with asymptomatic hyperamylasemia to combined immunotherapy of anti-cytotoxic T lymphocyte-associated antigen 4 plus anti-programmed cell death-1 antibodies.docx

BackgroundCombined immunotherapy has shown promising results in the treatment of advanced HCC, whereas the priority population that would respond to the combined immunotherapy is still elusive. In addition, HCC with asymptomatic hyperamylasemia was not reported previously.Case presentationAn aged patient was diagnosed as HCC with BCLC stage C (bone metastasis). Notably, this patient showed asymptomatic hyperamylasemia. The patient was then enrolled in a trial evaluating combined immunotherapy of anti-PD-1 antibody sintilimab (IBI308) plus anti-CTLA-4 antibody (IBI310) in advanced HCC. After being treated with combined immunotherapy, this patient rapidly achieved complete response (CR) according to mRECIST criteria or immune partial response (iPR) according to iRECIST criteria and maintain the CR state for more than 12 months. Interestingly, serum levels of amylase and lipase in this patient were reduced after treatment.ConclusionWe reported, for the first time, a case of metastatic HCC with asymptomatic hyperamylasemia, and suggested that HCC patients with asymptomatic hyperamylasemia may benefit from combined immunotherapy of anti-CTLA-4 and PD-1 antibodies.</p

11βHSD2 inhibition augmented corticosterone (CS)-induced COX-2 inhibition in lung cancer cells.

<p><b>A</b>. Immunoblotting indicated that 11βHSD2 was expressed in all investigated cell lines while COX-2 was detectable in some of them, including LLC, H1435 and A549 cells. <b>B</b>. Immunoblotting indicated that CS inhibited LLC cell COX-2 expression in a dose-dependent manner. <b>C</b>. CS (1μM)-induced LLC cell COX-2 inhibition was enhanced by 11βHSD2 inhibitor, GA. <b>D</b>. CS (1μM)-induced LLC cell COX-2 inhibition was enhanced by carbenoxolone (CBX, 10 μM), another 11βHSD2 inhibitor. <b>E</b>. CS (1μM)-induced H1435 cell COX-2 inhibition was enhanced by GA (10 μM). <b>F</b>. CS (1μM)-induced A549 cell COX-2 inhibition was enhanced by GA (10 μM).</p

Proposed mechanism underlying 11ßHSD2 activity and lung tumorigenesis.

<p>11ßHSD2 inhibition leads to increased levels of tumor intracellular active glucocorticoid and activation of glucocorticoid receptors. The subsequent inhibition of the COX-2, ERK and mTOR pathways leads to suppression of lung tumorigenesis.</p

11βHSD2 inhibition suppressed lung tumorigenesis.

<p><b>A</b>. LLC tumor growth was significantly attenuated by 11βHSD2 inhibition with GA (**P < 0.01, n = 8 in each group). LLC cell suspensions (100 μl, 5 x 10⁵ cells) were injected subcutaneously into the flank of C57/B6 mouse (2 sites). The mice were sacrificed 18 days later and tumor growth (tumor weight from two sites) was evaluated. <b>B</b>. Kaplan-Meier survival curve indicated that 11βHSD2 inhibition with GA increased survival probability in mice with tail vein injections of LLC cells (100 μl of LLC cell suspensions containing 5 x 10⁵ cells). ***P < 0.001, n = 12 in control and n = 14 in GA group. In both models, GA was given at 10 mg/kg/day (i.p. injection) starting one day before LLC cell injections.</p

11βHSD2 inhibition suppressed lung tumorigenesis in association with suppression of tumor ERK and mTOR activities in KrasLA2 mice.

<p><b>A</b>. GA treatment led to decreases in both number and size of lesion in lung surface of KrasLA2 mice (20 weeks of age). n = 9, **P < 0.01. <b>B</b>. Kaplan-Meier survival curve indicated that 11βHSD2 inhibition with GA increased survival probability of KrasLA2 mice. ***P < 0.001, n = 25 in vehicle group and n = 10 in GA group. <b>C</b>: GA treatment inhibited lung tumor levels of p-ERK. ***P < 0.001 vs. control, n = 4. Original magnification: x 160. <b>D</b>. GA treatment inhibited tumor p-mTOR expression. Original magnification: x 250. <b>E</b>. GA treatment inhibited tumor proliferation as indicated by decreased Ki67 positive cells in tumors. ***P < 0.001 vs. control, n = 4. Original magnification: x 400.</p

CS-induced inhibition of mTOR and ERK signaling pathways and cell proliferation was enhanced by 11βHSD2 inhibition with GA in lung cancer cell lines.

<p><b>(A and B)</b>. CS treatment led to decreased expression levels of p-mTOR and p-ERK in LLC cells <b>(A)</b> and A549 cells <b>(B)</b>, which were augmented by GA treatment. <b>(C and D)</b>. CS treatment led to inhibition of LLC <b>(C)</b> and A549 <b>(D)</b> cell proliferation in a dose-dependent manner, which was enhanced by GA treatment. ***P < 0.001, n = 3.</p

11βHSD2 inhibition with GA increased lung corticosterone levels in association with attenuation of tumor COX-2 expression in KrasLA2 mice.

<p><b>A</b>. GA treatment markedly increased lung active corticosterone levels and decreased inactive 11-keto-corticosterone levels in KrasLA2 mice (20 weeks of age). *P <0.05 vs. control, n = 6. <b>B</b>. Lung tumor COX-2 expression was suppressed by 11ßHSD2 inhibition with GA. **P < 0.01 vs. control, n = 3. Immunostaining showed markedly decreased tumor COX-2 with GA treatment. Original magnification: x 250. <b>C</b>. GA treatment increased mannose receptor (MR, CD206) expressing macrophages in tumor, an indication of increased tumor levels of active corticosterone. ***P < 0.001 vs. control, n = 4. Original magnification: x 160.</p

11ßHSD2 expression in mouse and human lung tumors.

<p><b>A</b> and <b>B</b>. Representative photomicrographs indicated 11ßHSD2 expression in small airway and alveolar epithelial cells of athymic nude mice <b>(A)</b> and in A549 lung tumor from athymic nude mice <b>(B)</b>. <b>C</b>. Representative photomicrographs showed that 11ßHSD2 immunostaining was strong in lung adenocarcinoma and squamous cell carcinoma, moderate in papillary carcinoma and small cell lung cancer (SCLC), but very weak in uninvolved lung tissue. Original magnification: x 160 in all.</p

Additional file 1 of HGF-mediated elevation of ETV1 facilitates hepatocellular carcinoma metastasis through upregulating PTK2 and c-MET

Additional file 1: Supplementary materials. FigureS1. (A) ETV1expression in LIHC and correlation of ETV1 expression with overallsurvival were analyzed in LIHC according to the data of The Cancer Genome Atlas(TCGA). (B) CellCounting Kit-8 (CCK8) assay assessing the cell proliferation of theETV1-overexpressing PLC/PRF/5 cells and ETV1-knockdown MHCC97H cells. (C) Colony formation assay showing the proliferationof the indicated HCC cells. Therepresentative photos were shown and the cell numbers were quantified. (D-F) Tumorgrowth of the indicated HCC cells was assessed by subcutaneous xenograft tumormodels. The tumor volume and weight were shown in (D) and (E), the representative images of Ki67 were shownin (F). n = 5 in each group. (G) The correlation between ETV1 expression and PTK2 or MET expression inTCGA-LIHC and GEO database. *p < 0.05, ****p < 0.0001. Data were shown as Mean ± SD. Figure S2. ETV1 binding sites withinthe promoter regions of PTK2. Thesequences highlighted in yellow represent the three binding sites of ETV1 onthe PTK2 promoter, and the arrow represents the transcription initiation sites.The   mutagenesis of the promoter sequencewere annotated. Figure S3. ETV1binding sites within the promoter regions of MET. The sequenceshighlighted in yellow represent the four binding sites of ETV1 onthe MET promoter, and the arrow represents the transcription initiation sites.The mutagenesis of the promoter sequence were annotated. Figure S4. (A-C) Western blotverifying PTK2 and MET knockdown effect in PLC/PRF/5-ETV1 cells and ELK1knockdown effect in PLC/PRF/5 cells. FigureS5. (A) The expression levels of MTDH, RHOA, TCF4 and MCL1 were determined in the indicated cells by real-time PCR. (B) Westernblotting assays of MTDH, RHOA, TCF4 and MCL1 in the indicated cells transfected with lentivirus. (C) The migratingand invasive capability of the indicated cells was determined via transwellassay. Figure S6. (A) The level of ETV1 in the PLC/PRF/5 cells upon HGFtreatment with/without ERK1/2 knockdown. Figure S7. Transcription factors binding siteswithin the promoter regions of ETV1. The sequences highlighted in blue represent the four binding sites ofELK1 on the ETV1 promoter. The yellow highlighted sequences representthe one binding site of ETS1 onthe ETV1 promoter. The sequenceshighlighted in grey represent the binding site of SP1 on the ETV1promoter. The red highlighted sequences represent the binding site of NF-ΚB1 onthe ETV1 promoter. The pink highlighted sequences represent the bindingsequence of STAT3 on the ETV1 promoter. The arrows representtranscription start sites. The mutagenesis of the promoter sequence wereannotated. Figure S8. (A)Representative IHC staining of ETV1 is shown. (B) Pearsoncorrelation analyses between ETV1 IHC score and the levels of serum HGF in HCCpatients. n=30. (C) The correlation between ETV1 expression and HGF expression inTCGA-LIHC and GEO database.(D) The correlation between ETV1expression and ELK1 expression in TCGA-LIHC and GEO database. Figure S9. Effectof ERK1/2 inhibitor on HGF-mediated HCC cell migration and invasion. (A)Transwell assays displayed the migratory and invasive capacity of theindicated cells upon HGF treatment. (B) Transwell assays displayed themigratory and invasive capacity of the indicated cells. Supplementary Table S1. List of genesdifferentially expressed in PLC/PRF/5-ETV1 versus PLC/PRF/5-Control cells usinga human liver cancer PCR array. Supplementary Table S2. List of genes differentially expressed in MHCC97H-shETV1 versusMHCC97H-shControl cells using a human liver cancer PCR array. Supplementary Table S3. Primer sequences usedin the study. Supplementary Table S4. Knockdown shRNA sequencesused in this study. Supplementary Table S5. Correlation between PTK2 expression and clinicopathologicalcharacteristics of HCCs in two independent cohorts of human HCC tissues. Supplementary Table S6. Correlationbetween c-MET expression and clinicopathological characteristics of HCCs in twoindependent cohorts of human HCC tissues

DataSheet_1_Identification of necroptosis-related subtypes, development of a novel signature, and characterization of immune infiltration in colorectal cancer.docx

IntroductionNecroptosis, a type of programmed cell death, has recently been extensively studied as an important pathway regulating tumor development, metastasis, and immunity. However, the expression patterns of necroptosis-related genes (NRGs) in colorectal cancer (CRC) and their potential roles in the tumor microenvironment (TME) have not been elucidated.MethodsWe explored the expression patterns of NRGs in 1247 colorectal cancer samples from genetics and transcriptional perspective. Based on a consensus clustering algorithm, we identified NRG molecular subtypes and gene subtypes, respectively. Furthermore, we constructed a necroptosis-related signature for predicting overall survival time and verified the predictive ability of the model. Using the ESTIMATE, CIBERSORT, and ssGSEA algorithms, we assessed the association between the above subtypes, scores and immune infiltration. ResultsMost NRGs were differentially expressed between CRC tissues and normal tissues. We found that distinct subtypes exhibited different NRGs expression, patients’ prognosis, immune checkpoint gene expression, and immune infiltration characteristics. The scores calculated from the necroptosis-related signature can be used to classify patients into high-risk and low-risk groups, with the high-risk group corresponding to reduced immune cell infiltration and immune function, and a greater risk of immune dysfunction and immune escape. DiscussionOur comprehensive analysis of NRGs in CRC demonstrated their potential role in clinicopathological features, prognosis, and immune infiltration in the TME. These findings help us deepen our understanding of NRGs and the tumor microenvironment landscape, and lay a foundation for effectively assessing patient outcomes and promoting more effective immunotherapy.</p