Supplementary Material for: Correlation of Neuroendocrine Differentiation with A Distinctively Suppressive Immune Microenvironment in Gastric Cancer

Introduction Neuroendocrine neoplasms (NENs) harbored significantly suppressive tumor immune microenvironments (TIMEs). However, the immunological effects of neuroendocrine differentiation (NED) on non-NE neoplasms, such as gastric cancer (GC), were unknown. Methods Between pure gastric cancer (PGC) and GC-NED, TIME features were scored based on expression data and validated on serial whole-tissue sections of surgical samples, with tertiary lymphoid structures (TLSs) and the extra-TLS zone evaluated independently using multi-marker immunohistochemical staining. Risk analyses of TIME features on tumor behaviors were performed in GC-NED. The universal immunological effects of NED were explored preliminarily in adenocarcinomas arising in other organs. Results Based on over 11,500 annotated TLSs and 2,700 extra-TLS zones, compared with PGC, GC-NED harbored a distinctively more suppressive TIME characterized by increased but immature TLSs, with higher naïve B cell and follicular regulatory T cell densities and downregulated TLS maturation-related cell ratios inside TLSs; increased naïve B cell and regulatory T cell densities and a high proportion of exhausted T cells in the extra-TLS zone. The upregulated tumor PD-L1 expression and its close correlations with TLS formation and maturation were remarkable exclusively in GC-NED. TIME features, especially those regarding TLSs, were significantly correlated with tumor growth and invasion. The desynchrony between TLS formation and maturation and increased naïve or regulatory immune cell infiltration was observed in adenocarcinomas of the colorectum, pancreas, lung, and prostate. Conclusion NED highlighted a distinct GC entity with more suppressive TIME features correlated with tumor behaviors, indicating a cohort that would benefit more from immunotherapies

Supplementary Material for: Correlation of Neuroendocrine Differentiation with A Distinctively Suppressive Immune Microenvironment in Gastric Cancer

Introduction Neuroendocrine neoplasms (NENs) harbored significantly suppressive tumor immune microenvironments (TIMEs). However, the immunological effects of neuroendocrine differentiation (NED) on non-NE neoplasms, such as gastric cancer (GC), were unknown. Methods Between pure gastric cancer (PGC) and GC-NED, TIME features were scored based on expression data and validated on serial whole-tissue sections of surgical samples, with tertiary lymphoid structures (TLSs) and the extra-TLS zone evaluated independently using multi-marker immunohistochemical staining. Risk analyses of TIME features on tumor behaviors were performed in GC-NED. The universal immunological effects of NED were explored preliminarily in adenocarcinomas arising in other organs. Results Based on over 11,500 annotated TLSs and 2,700 extra-TLS zones, compared with PGC, GC-NED harbored a distinctively more suppressive TIME characterized by increased but immature TLSs, with higher naïve B cell and follicular regulatory T cell densities and downregulated TLS maturation-related cell ratios inside TLSs; increased naïve B cell and regulatory T cell densities and a high proportion of exhausted T cells in the extra-TLS zone. The upregulated tumor PD-L1 expression and its close correlations with TLS formation and maturation were remarkable exclusively in GC-NED. TIME features, especially those regarding TLSs, were significantly correlated with tumor growth and invasion. The desynchrony between TLS formation and maturation and increased naïve or regulatory immune cell infiltration was observed in adenocarcinomas of the colorectum, pancreas, lung, and prostate. Conclusion NED highlighted a distinct GC entity with more suppressive TIME features correlated with tumor behaviors, indicating a cohort that would benefit more from immunotherapies

Supplementary Material for: Correlation of Neuroendocrine Differentiation with A Distinctively Suppressive Immune Microenvironment in Gastric Cancer

Introduction Neuroendocrine neoplasms (NENs) harbored significantly suppressive tumor immune microenvironments (TIMEs). However, the immunological effects of neuroendocrine differentiation (NED) on non-NE neoplasms, such as gastric cancer (GC), were unknown. Methods Between pure gastric cancer (PGC) and GC-NED, TIME features were scored based on expression data and validated on serial whole-tissue sections of surgical samples, with tertiary lymphoid structures (TLSs) and the extra-TLS zone evaluated independently using multi-marker immunohistochemical staining. Risk analyses of TIME features on tumor behaviors were performed in GC-NED. The universal immunological effects of NED were explored preliminarily in adenocarcinomas arising in other organs. Results Based on over 11,500 annotated TLSs and 2,700 extra-TLS zones, compared with PGC, GC-NED harbored a distinctively more suppressive TIME characterized by increased but immature TLSs, with higher naïve B cell and follicular regulatory T cell densities and downregulated TLS maturation-related cell ratios inside TLSs; increased naïve B cell and regulatory T cell densities and a high proportion of exhausted T cells in the extra-TLS zone. The upregulated tumor PD-L1 expression and its close correlations with TLS formation and maturation were remarkable exclusively in GC-NED. TIME features, especially those regarding TLSs, were significantly correlated with tumor growth and invasion. The desynchrony between TLS formation and maturation and increased naïve or regulatory immune cell infiltration was observed in adenocarcinomas of the colorectum, pancreas, lung, and prostate. Conclusion NED highlighted a distinct GC entity with more suppressive TIME features correlated with tumor behaviors, indicating a cohort that would benefit more from immunotherapies

Supplementary Material for: Integrated High Throughput Analysis Identifies GSK3 as a Crucial Determinant of p53-Mediated Apoptosis in Lung Cancer Cells

<strong><em>Background/Aims:</em></strong> p53 dysfunction is frequently observed in lung cancer. Although restoring the tumour suppressor function of p53 is recently approved as a putative strategy for combating cancers, the lack of understanding of the molecular mechanism underlying p53-mediated lung cancer suppression has limited the application of p53-based therapies in lung cancer. <b><i>Methods and Results:</i></b> Using RNA sequencing, we determined the transcriptional profile of human non-small cell lung carcinoma A549 cells after treatment with two p53-activating chemical compounds, nutlin and RITA, which could induce A549 cell cycle arrest and apoptosis, respectively. Bioinformatics analysis of genome-wide gene expression data showed that distinct transcription profiles were induced by nutlin and RITA and 66 pathways were differentially regulated by these two compounds. However, only two of these pathways, 'Adherens junction' and 'Axon guidance', were found to be synthetic lethal with p53 re-activation, as determined via integrated analysis of genome-wide gene expression profile and short hairpin RNA (shRNA) screening. Further functional protein association analysis of significantly regulated genes associated with these two synthetic lethal pathways indicated that GSK3 played a key role in p53-mediated A549 cell apoptosis, and then gene function study was performed, which revealed that GSK3 inhibition promoted p53-mediated A549 cell apoptosis in a p53 post-translational activity-dependent manner. <b><i>Conclusion:</i></b> Our findings provide us with new insights regarding the mechanism by which p53 mediates A549 apoptosis and may cast light on the development of more efficient p53-based strategies for treating lung cancer