In vitro CRISPR screening uncovers CRTC3 as a regulator of IFN-γ-induced ferroptosis of hepatocellular carcinoma.

Interferon-gamma (IFN-γ) exerts anti-tumor effects by inducing ferroptosis. Based on CRISPR/Cas9 knockout screening targeting genome-wide protein encoding genes in HepG2 and SK-Hep-1 cell lines, we found that cAMP response element-binding protein (CREB) regulated transcription coactivator 3 (CRTC3) protects tumor cells from drug-induced ferroptosis and significantly inhibits the efficacy of IFN-γ treatment in hepatocellular carcinoma (HCC). Mechanistically, CRTC3 knockout altered tumor cell lipid patterns and increased the abundance of polyunsaturated fatty acids (PUFAs), which enables lipid peroxidation and enhances the susceptibility of HCC cells to ferroptosis inducers. To scavenge for accumulated lipid peroxides (LPO) and maintain redox equilibrium, HCC cells up-regulate SLC7A11 and glutathione peroxidase 4 (GPx4) expressions to enhance the activities of glutamate-cystine antiporter (system xc-) and LPO clearance. As IFN-γ inhibiting system xc-, simultaneous treatment with IFN-γ disrupts the compensatory mechanism, and generates a synergistic effect with CRTC3 knockout to facilitate ferroptosis. Sensitizing effects of CRTC3 depletion were confirmed using typical ferroptosis inducers, including RSL3 and erastin. Sorafeinib, a commonly used target drug in HCC, was repeatedly reported as a ferroptosis inducer. We then conducted both in vitro and vivo experiments and demonstrated that CRTC3 depletion sensitized HCC cells to sorafenib treatment. In conclusion, CRTC3 is involved in the regulation of PUFAs metabolism and ferroptosis. Targeting CRTC3 signaling in combination with ferroptosis inducers present a viable approach for HCC treatment and overcoming drug resistance.

TP53 Exon 5 Mutation Indicates Poor Progression-Free Survival for Patients with Stage IV NSCLC.

BACKGROUND: Genetic mutations are quite common in non-small cell lung cancer (NSCLC), however, their prognostic value remains controversial. METHODS: This study explored the mutational landscape of tumor samples from patients with advanced NSCLC by next-generation sequencing (NGS). A total of 101 NSCLC patients in stage III or IV receiving first-line treatment were included. RESULTS: TP53 mutation was the most frequent genetic alteration in NSCLC tumors (68%), followed by EGFR (49%), CDKN2A (12%), LRP1B (9%), and FAT3 (9%) mutations. Among 85 patients with stage IV NSCLC, first-line targeted therapy remarkably prolonged progression-free survival (PFS) of patients compared with first-line chemotherapy (p = 0.0028). Among 65 patients with stage IV NSCLC whose tumors harbored EGFR, ALK, ROS, or BRAF mutations, first-line targeted therapy substantially prolonged the PFS of patients (p = 0.0027). In patients with TP53 mutations who received first-line targeted therapy or chemotherapy, missense mutation was the most common mutation type (36/78), and exon 5 represented the most common mutated site (16/78). CONCLUSIONS: TP53 mutation in exon 5 could independently predict poor PFS of patients with stage IV NSCLC after the first- line treatment. Moreover, mutations in TP53 exon 5 and LRP1B were associated with shorter PFS of such patients whether after first-line chemotherapy or targeted therapy, respectively. Thus, these patients should be given immunotherapy or immunochemotherapy.

Spatial distribution of tumor-infiltrating T cells indicated immune response status under chemoradiotherapy plus PD-1 blockade in esophageal cancer.

Background: The spatial distribution of tumor-infiltrating T cells and its dynamics during chemoradiotherapy combined with PD-1 blockade is little known in esophageal squamous cell carcinoma (ESCC). Methods: We applied the multiplex immunofluorescence method to identify T cells (CD4+, CD8+ T cells, and their PD-1- or PD-1+ subsets) and myeloid-derived cells (CD11c+ dendritic cells, CD68+ macrophages, and their PD-L1+ subpopulations) in paired tumor biopsies (n = 36) collected at baseline and during combination (40 Gy of radiation) from a phase Ib trial (NCT03671265) of ESCC patients treated with first-line chemoradiotherapy plus anti-PD-1 antibody camrelizumab. We used the FoundationOne CDx assay to evaluate tumor mutational burden (TMB) in baseline tumor biopsies (n = 14). We dynamically assessed the nearest distance and proximity of T-cell subsets to tumor cells under combination and estimated the association between T-cell spatial distribution and combination outcome, myeloid-derived subsets, TMB, and patient baseline characteristics. Findings: We found that the tumor compartment had lower T-cell subsets than the stromal compartment but maintained a comparable level under combination. Both before and under combination, PD-1- T cells were located closer than PD-1+ T cells to tumor cells; T cells, dendritic cells, and macrophages showed the highest accumulation in the 5-10-µm distance. Higher CD4+ T cells in the tumor compartment and a shorter nearest distance of T-cell subsets at baseline predicted poor OS. Higher baseline CD4+ T cells, dendritic cells, and macrophages were associated with worse OS in less than 10-µm distance to tumor cells, but related with better OS in the farther distance. Higher on-treatment PD-1-positive-expressed CD4+ and CD8+ T cells within the 100-µm distance to tumor cells predicted longer OS. T cells, dendritic cells, and macrophages showed a positive spatial correlation. Both high TMB and smoking history were associated with a closer location of T cells to tumor cells at baseline. Conclusions: We firstly illustrated the T-cell spatial distribution in ESCC. Combining chemoradiotherapy with PD-1 blockade could improve the antitumor immune microenvironment, which benefits the treatment outcome. Further understanding the precision spatiality of tumor-infiltrating T cells would provide new evidence for the tumor immune microenvironment and for the combination treatment with immunotherapy.

Radiotherapy plus camrelizumab affects peripheral CD8 T-cell differentiation subsets expressing PD-1, TIGIT, and CTLA-4 in esophageal squamous cell carcinoma.

Our previous phase Ib trial (NCT03222440) showed that radiotherapy plus the anti-PD-1 antibody camrelizumab is a safe and feasible first-line therapy for locally advanced esophageal squamous cell carcinoma. In this study, we divided peripheral CD8 T-cell differentiation subsets into 4 subpopulations (naive T cells, central memory T cells, effector memory T cells, and CD45RA+ effector memory T cells). We then investigated the influence of radiotherapy plus camrelizumab therapy on the proportions of the 4 subsets and their PD-1, TIGIT, and CTLA-4 expression as well as their proliferative activity and compared the effects with those of concurrent chemoradiotherapy. Nineteen and 15 patients with esophageal squamous cell carcinoma who received radiotherapy plus camrelizumab therapy and concurrent chemoradiotherapy, respectively, were enrolled in this study. We isolated peripheral blood mononuclear cells from these patients before treatment and longitudinally after the delivery of 40 Gy radiotherapy. Flow cytometry was conducted to detect peripheral CD8 T-cell subsets and PD-1, TIGIT, CTLA-4, and Ki67 expression levels in patients with esophageal squamous cell carcinoma. We found that radiotherapy plus camrelizumab therapy did not change the proportions of the 4 subsets or the expression of CTLA-4, but this therapy decreased PD-1 expression by the 4 subsets and TIGIT expression by effector memory T cells, as well as significantly enhanced the proliferative activity of CD8 T cells, whereas concurrent chemoradiotherapy produced different effects. In addition, we further identified peripheral biomarkers that potentially predict the outcome of radiotherapy plus camrelizumab therapy.

Comparison of dynamic changes in the peripheral CD8+ T cells function and differentiation in ESCC patients treated with radiotherapy combined with anti-PD-1 antibody or concurrent chemoradiotherapy.

Objective: The systematic immune status of cancer patients undergoing immunotherapy is little known. We prospectively identified the function and differentiation traits of peripheral CD8+ T cells based on our phase 1b clinical trial (NCT03222440) of radiotherapy combined with camrelizumab in patients with locally advanced esophageal squamous cell carcinoma (ESCC) and compared it with concurrent chemoradiotherapy (CCRT). Methods: 19 and 18 patients were included in the cohort of radiotherapy plus camrelizumab and cohort of CCRT treatment. By using flow cytometry, we evaluated the expression levels of PD-1, Eomes, T-bet and IFN-γ (function), CD38 and HLA-DR (activation), and differentiation subsets classified according to the expression levels of CD45RA and CD62L in peripheral CD8+ T cells before and during treatment. Results: Effective binding of anti-PD-1 antibody camrelizumab with PD-1 on CD8+ T cells was detected during treatment. Both two treatments elevated the expression levels of activation molecules CD38 and HLA-DR on CD8+ T cells. PD-1+CD8+ T cells had more activation features than PD-1-CD8+ T cells in two groups and the treatments did not alter these differences. The two treatments activated both PD-1+ and PD-1- CD8+ T cells. PD-1+CD8+ T cells had less Naïve and TEMRA but more Tcm and Tem than PD-1-CD8+ T cells in two groups and both two treatments changed the ratio of memory T cells in PD-1+ and PD-1- cells. RT plus camrelizumab treatment reduced Naïve T cells and TEMRA subsets both in PD-1+ and PD-1- CD8+ T cells while elevated Tcm subset in PD-1+CD8+ T cells and Tem subset in PD-1-CD8+ T cells. CCRT elevated Tcm subset and reduced TEMRA subset in PD-1-CD8+ T cells while did not change any subset in PD-1+CD8+ T cells. Furthermore, patients undergoing radiotherapy plus immunotherapy were found to obtain better prognosis than those receiving CCRT. Conclusions: This study identified the dynamic changes of systematic immune status of patients undergoing treatment. The two treatments had similar activation effects on peripheral CD8+ T cells with different PD-1 properties but had different effects on their differentiation status. These results provided potential clues to the reasons underlying the difference in prognosis of the two treatments.

Single-cell profiling of immune cells after neoadjuvant pembrolizumab and chemotherapy in IIIA non-small cell lung cancer (NSCLC).

The combination of immune checkpoint inhibitors (ICIs) with chemotherapy (chemoimmunotherapy) in the neoadjuvant setting have achieved favorable clinical benefits in non-small cell lung cancer (NSCLC), but the mechanism of clinical responses remain unclear. We provide a rich resource of 186,477 individual immune cells from 48 samples of four treatment-naive and eight neoadjuvant chemoimmunotherapy treated IIIA NSCLC patients (responders versus non-responders) by single-cell RNA-seq and TCR-seq. We observed the synergistic increase of B cells and CD4+ T cells were associated with a positive therapeutic response of neoadjuvant chemoimmunotherapy. B cell IgG subclasses IgG1 and IgG3 played a critical role in anti-tumor immune response in tumor lesions, and this process was driven by increased IL-21 secreted by infiltrated T follicular helper (Tfh) cells after neoadjuvant chemoimmunotherapy. Furthermore, we uncovered several critical events for positive clinical outcomes, including the diminished activated TNFRSF4+ regulatory T cells (Tregs), increased LAMP3+ dendritic cells (DCs), and the expansion of intratumoral CD4+ T clones and peripheral C3-Cytotoxic CD8+ T clones. A validation cohort of 26 treatment-naive and 30 neoadjuvant chemoimmunotherapy treated IIIA/ IIIB NSCLC patients verified these findings. In total, our comprehensive study of the single-cell profile of immune cells provides insights into mechanisms underlying anti-PD-1-based therapies and identified potential predictive factors and therapeutic targets for improving the efficiency of neoadjuvant chemoimmunotherapy in NSCLC.

Dysregulated Th1 cells in lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is a common subtype of lung cancer. Th1 cells contribute to antitumor immune responses. However, there are few studies on Th1 cells in LUSC. CD8+ T cells are the main driver of the antitumor immunity, targeting tumor cells killing. Th1 cells play an important auxiliary role in this process. Here, we used single-cell RNA-seq (scRNA-seq) to analyze qualified CD4+ T cells and Th1 cells (defined CD4+ T cells with 1 or more of STAT1+ , STAT4+ , T-bet+ , and IFN-γ+ as Th1 cells) from tissues of 8 LUSC patients. Then, we validated Th1 cells and CD8+ T cells of 32 LUSC patients by multiplex immunofluorescence staining and immunohistochemistry. Finally, we used flow cytometry to detect IFN-γ of CD4+ T cells in human PBMCs coincubated with LUSC-derived supernatant to simulate a tumor inhibitory microenvironment. ScRNA-seq showed IFN-γ+ Th1 cells account for 25.28% of all Th1 cells. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses of differentially expressed genes between IFN-γ+ Th1 cells and IFN-γ- Th1 cells confirmed the decreased IFN-γ is associated with endoplasmic reticulum stress (ER stress). Multiplex immunofluorescence staining and immunohistochemistry proved there was a positive correlation between IFN-γ+ STAT1+ T-bet+ Th1 cells and CD8+ T cells. Flow cytometry showed IFN-γ secreted by Th1 cells is decreased. These findings support the claim that Th1 cells' function is suppressed in LUSC. Through scRNA-seq, we found that the decreased Th1 cells' function is associated with ER stress, which requires further study. Overall, these findings may produce a new method for the treatment of LUSC.

Time-spatial analysis of T cell receptor repertoire in esophageal squamous cell carcinoma patients treated with combined radiotherapy and PD-1 blockade.

T cell receptor (TCR) repertoire as a biomarker for predicting immunotherapy efficiency has been widely studied. However, its dynamics during radiotherapy combined with PD-1 blockade is little known. Using paired tumor and blood samples from the phase Ib clinical study (NCT03222440), we investigate the time-spatial TCR repertoire in esophageal squamous cell carcinoma (ESCC) patients treated with first-line definitive radiotherapy concurrently with anti-PD-1 antibody camrelizumab, and also evaluate the association between TCR repertoire and clinical outcomes. TCR sequencing was performed on tumor biopsies (n = 34, 15 pairs) and peripheral CD8+ T cells (n = 36, 18 pairs) collected at baseline and during treatment (after 40 Gy radiation and 2 rounds of camrelizumab). Whole exome sequencing was applied to estimate genomic mutations and tumor mutation burden. We show that the intratumoral TCR repertoire at baseline was correlated with tumor microenvironment and presented heterogeneity inter-individually. T-cell clones inflowed mutually between tumors and peripheral blood under combination treatment, resulting in an elevation of intratumoral TCR diversity. The peripheral CD8+ TCR diversity at baseline, increased tumor-peripheral Morisita-Horn overlap during treatment, and expansion of persistent intratumoral T-cell clones during treatment predicted improved survival. While it is unclear whether radiation contributed to the TCR changes versus PD-1 therapy alone, our results firstly reveal radiotherapy combined with PD-1 blockade greatly promoted time-spatial alteration of TCR repertoire between tumor and peripheral blood, which demonstrate the peripheral CD8+ TCR diversity at baseline and dynamic alteration of intratumoral TCRs acted as potential effective biomarkers of radiotherapy combined with immunotherapy in ESCC.

Addition of camrelizumab to docetaxel, cisplatin, and radiation therapy in patients with locally advanced esophageal squamous cell carcinoma: a phase 1b study.

Patients with locally advanced esophageal squamous cell carcinoma (ESCC) show poor survival after concurrent chemoradiotherapy. This study investigated the safety and feasibility of combining concurrent chemoradiotherapy with the anti-PD-1 antibody camrelizumab as first-line treatment for these patients. In this phase 1b study (ClinicalTrials.gov NCT03671265), patients received concurrent chemotherapy (cisplatin [25 mg/m2] plus docetaxel [25 mg/m2] for 4 weeks) and radiotherapy (2.0 Gy/fraction, total 60 Gy) with camrelizumab (200 mg every 2 weeks for 32 weeks). Primary endpoints were safety and tolerability, and health-related quality of life. Secondary endpoints were radiological and pathological response rates, overall survival (OS), and progression-free survival (PFS). Candidate biomarkers in tumor and peripheral blood were monitored at baseline and after 40 Gy radiation. Twenty patients were enrolled. The most common treatment-related grade 3 adverse events included radiation esophagitis (20%) and esophageal fistula (10%). Serious treatment-related adverse events occurred in eight (40%) patients. No treatment-related deaths were reported. Health-related quality of life did not deteriorate. Thirteen (65%) patients had an objective response after 40 Gy radiation. At a median follow-up of 23.7 months (95% CI 21.9-24.5), OS and PFS time ranged from 8.2-28.5 and 4.0-28.5 months, respectively. The 12-month and 24-month OS rate was 85.0% and 69.6%; PFS rate was 80.0% and 65.0%. Tumor PD-L1 expression and CD11c+ dendritic cells and peripheral-blood IL-27, IL-15, Eotaxin-3, and IL-22 were associated with OS. First-line concurrent chemoradiotherapy plus camrelizumab had a manageable safety profile and promising antitumour efficacy for ESCC, and deserves further study.

Safety and Feasibility of Radiotherapy Plus Camrelizumab for Locally Advanced Esophageal Squamous Cell Carcinoma.

LESSONS LEARNED: Radiotherapy plus anti-PD-1 antibody as first-line therapy is safe and feasible in locally advanced esophageal squamous cell carcinoma (ESCC). Tumor-infiltrating and peripheral lymphocytes were associated with patient survival. Further studies combining chemoradiotherapy with immunotherapy in locally advanced ESCC and exploration of predictive biomarkers are warranted. BACKGROUND: We conducted a phase Ib study of radiotherapy plus programmed cell death protein 1 (PD-1) monoclonal antibody camrelizumab as first-line treatment for locally advanced esophageal squamous cell carcinoma (ESCC). METHODS: We planned to enroll 20 patients with newly diagnosed locally advanced ESCC. Patients received 60 Gy radiation (2.0 Gy/fraction, 5 fractions/week), with camrelizumab (200 mg every 2 weeks) starting with radiotherapy and continuing for 32 weeks (i.e., for 16 cycles). The primary endpoints were safety and feasibility. Secondary endpoints were rates of radiologic and pathologic response, overall survival (OS), and progression-free survival (PFS). Study data were collected by the week during radiotherapy (RT), every month during the maintenance camrelizumab treatment, and every 3 months after treatment. Tumor microenvironment and peripheral blood were monitored at baseline and after 40 Gy radiation for association with efficacy. RESULTS: Twenty patients were enrolled and received treatment. One patient (patient 10) was excluded upon discovery of a second tumor in the bladder during treatment, leaving 19 patients for analysis. Toxicity was deemed tolerable. Fourteen (74%) patients had assessed objective response. At a median follow-up time of 31.0 months (95% confidence interval [CI], 27.0-35.1), median OS and PFS times were 16.7 months (95% CI, 5.9-27.9) and 11.7 months (95% CI, 0-30.3), respectively. OS and PFS rates at 24 months were 31.6% and 35.5%, respectively. Kaplan-Meier analysis revealed associations between the following factors and OS/PFS: tumor programmed cell death ligand 1 (PD-L1) expression, PD-1+ CD8+ , PD-1+ CD4+ T cells, and PD-L1+ CD4+ T cells; peripheral blood CD4+ , CD8+ , CD4+ regulatory T cells, and their subsets. CONCLUSION: Radiotherapy plus camrelizumab had manageable toxicity and antitumor efficacy for locally advanced ESCC. Several biomarkers were associated with clinical benefit and deserve further study.

Lung cancer-associated mesenchymal stem cells promote tumor metastasis and tumorigenesis by induction of epithelial-mesenchymal transition and stem-like reprogram.

Mesenchymal stem cells (MSCs) have attracted more attention in antitumor therapy by using MSCs as vehicles or targeting modulators of MSCs. But their role and mechanisms in tumor progression are less known. In the present study, we successfully isolated pairs of MSCs from lung cancer (LC-MSCs) and adjacent tumor-free tissues. Based on the coculture system in vitro and animal studies in vivo, we originally found that LC-MSCs significantly promoted tumor metastasis and tumorigenesis both in vitro and in vivo. Partial epithelial-mesenchymal transition (EMT) was induced in lung cancer cells by LC-MSCs by the evidence of remarkable increase in snail and slug expression but not in other EMT-associated genes. The expression of stem related genes also escalated significantly. And spheroids perfectly formed when tumor cells were co-incubated with LC-MSCs. These results revealed a close link of partial EMT and acquisition of stem-like traits in lung cancer cells which was induced by LC-MSCs and greatly promoted metastasis and tumorigenesis in lung cancer. Our findings provided a new insight into LC-MSCs in tumor progression and helped to identify LC-MSCs as a potential vehicle or target for lung cancer therapy.

Spatial Distribution and Predictive Significance of Dendritic Cells and Macrophages in Esophageal Cancer Treated With Combined Chemoradiotherapy and PD-1 Blockade.

Background: The first clinical study (NCT03671265) of first-line chemoradiotherapy combined with PD-1 blockade showed promising treatment outcomes in locally advanced esophageal squamous cell carcinoma (ESCC). However, partial patients did not respond to the combination treatment. The roles of dendritic cells (DCs) and macrophages in this combination treatment remain poorly understood. Methods: We performed multiplexed immunofluorescence method to identify CD11c+ DCs, CD68+ macrophages, and their PD-L1- or PD-L1+ subpopulations in paired tumor biopsies (n = 36) collected at baseline and during the combination treatment (after radiation, 40 Gy) from the phase Ib trial (NCT03671265). We applied whole exome sequencing in the baseline tumor biopsies (n = 14) to estimate tumor mutation burden (TMB). We dynamically investigated the spatial distribution of DCs and macrophages under chemoradiotherapy combined with PD-1 blockade, and evaluated the association between their spatial distribution and combination outcome, and TMB. Results: The results showed that high percentages of PD-L1- DCs and macrophages in the baseline tumor compartment, but not in the stromal compartment, predicted improved OS and PFS. Chemoradiotherapy combined with PD-1 blockade promoted DCs and macrophages to migrate closer to tumor cells. During combination treatment, PD-L1- tumor cells were nearest to PD-L1- DCs and macrophages, while PD-L1+ tumor cells were next to PD-L1+ DCs and macrophages. High TMB was closely associated with a shorter distance from tumor cells to DCs and macrophages. Shorter distance between PD-L1+ tumor cells and PD-L1+ DCs or PD-L1- macrophages during the combination was correlated with better OS. Shorter distance between PD-L1- tumor cells and PD-L1- macrophages during combination was associated with both longer OS and PFS. Conclusions: PD-L1- or PD-L1+ DCs and macrophages exhibit distinct spatial distribution in ESCC. The close distance between tumor cells and these antigen-presenting cells (APCs) is critical to the clinical outcome in chemoradiotherapy combined with PD-1 blockade in ESCC patients. Our results highlight the predictive potential of spatial patterns of APCs in chemoradiotherapy combined with immunotherapy and reveal the underlying mechanism of APCs participating in chemoradiotherapy-induced antitumor immune response in ESCC.

Memory stem T cells generated by Wnt signaling from blood of human renal clear cell carcinoma patients.

OBJECTIVE: Memory stem T cells (Tscm) have attracted attention because of their enhanced self-renewal, multipotent capacity, and anti-tumor capacities. However, little is known about Tscm in patients with renal clear cell carcinoma (RCC) and the role of Wnt signaling in these cells. We evaluated Tscm from RCC patients concerning their activation of Wnt signaling in vitro and explored the mechanism of preferential survival. METHODS: Flow cytometry identified surface markers and cytokines produced from accumulated Tscm in the presence of the glycogen synthase kinase beta inhibitor TWS119. Apoptosis was evaluated after induction using tumor necrosis factor-alpha. Immunofluorescence and Western blot analyses were used to investigate the activation of the nuclear factor-kappa B (NF-КB) pathway. RESULTS: RCC patients had a similar percentage of CD4+ and CD8+ Tscm as healthy donors. Activation of Wnt signaling by TWS119 resulted in the accumulation of Tscm in activated T cells, but reversal of differentiated T cells to Tscm was not achieved. Preferential survival of Tscm was associated with increased anti-apoptotic ability mediated downstream of the NF-КB activation pathway. CONCLUSIONS: The finding that Tscm can accumulate by Wnt signaling in vitro in blood from RCC patients will help in devising new cancer therapy strategies of Tscm-based adoptive immunotherapy, such as dendritic cell-stimulated Tscm, and T cell receptor or chimeric antigen receptor-engineered Tscm.

Chemoradiotherapy-Induced CD4+ and CD8+ T-Cell Alterations to Predict Patient Outcomes in Esophageal Squamous Cell Carcinoma.

Purpose and Objectives: Chemoradiotherapy (CRT) is an important component of treatment for patients with locally advanced esophageal squamous cell carcinoma (ESCC). Recent research findings support the role of CRT in activating an anti-tumor immune response. However, predictors of CRT efficacy are not fully understood. The aim of this study was to measure CRT-induced changes to lymphocyte subpopulations and to evaluate the prognostic value of lymphocyte alterations for patients with ESCC. Materials and Methods: In total, this pilot study enrolled 64 patients with ESCC who received neo-adjuvant CRT or definitive CRT. Peripheral blood samples were collected before and during treatment and were analyzed by flow cytometry for CD19, CD3, CD4, CD8, CD56, and CD16. Relationships between lymphocyte subset alterations and overall survival (OS) and progression-free survival (PFS) were evaluated using the log-rank test and a Cox regression model. Results: The median follow-up period was 11.8 months (range, 4.0-20.2 months). Compared to pre-treatment specimens, post-treatment blood samples had decreased proportions of CD19+ B-cells and increased proportions of CD3+ and CD8+ T-cells (all P < 0.05). Univariate and multivariate analysis showed that increased CD4+ T-cell ratios after CRT independently predicted superior PFS (hazard ratio [HR] = 0.383; 95% confidence interval [CI] = 0.173-0.848, P = 0.017) and that increased CD8+ T-cell ratios predicted improved OS (HR = 0.258; 95% CI = 0.083-0.802, P = 0.019). Patients with both increased CD4+ and CD8+ ratios had a superior PFS and OS, compared to patients with an increased CD4+ ratio only or CD8+ ratio only or neither (1-year PFS rate 63 vs. 25%, 1-year OS rate 80 vs. 62%, P = 0.005 and 0.025, respectively). Conclusions: CRT-induced increases in CD4+ and CD8+ T-cell ratios are reliable biomarker predictors of survival in patients with ESCC.

Identification of prognosis-related genes in hepatocellular carcinoma based on bioinformatical analysis / 中国肿瘤生物治疗杂志

@# Objective: To identify the differentially expressed genes (DEGs) between hepatocellular carcinoma (HCC) tissues and normal liver tissues by bioinformatic methods, and to explore the intrinsic mechanism of these candidate genes involving in the occurrence and development of HCC from transcriptome level as well as the clinical significance of their associations with the prognosis of HCC patients. Methods: Gene expression profiles of GSE45267, GSE64041, GSE84402 and TCGA were downloaded from GEO (Gene Expression Omnibus) and TCGA(The Cancer GenomeAtlas), respectively. R software and Bioconductor packages were used to identify the DEGs between HCC tissues and para-cancer tissues, and then Gene Ontology (GO) Enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Protein-Protein Interaction (PPI) network analysis and survival analysis were performed. Results: Forty-six up-regulated genes and 154 down-regulated genes were screened out,and GO enrichment analysis showed that these DEGs were mainly related to cell division, proliferation, cycle regulation, oxidation-reduction process and certain metabolic pathways. KEGG pathway analysis revealed that DEGs were mainly involved in tryptophan metabolism, retinol metabolism and other metabolic pathways as well as p53 pathway. Over-expression of a panel of up-regulated genes (CCNA2, CDK1, DLGAP5, KIF20A, KPNA2 and MELK) was shown to be significantly negatively correlated with the prognosis of HCC patients in the TCGA dataset (all P<0.01). Conclusion: A set of up-regulated hub genes that are negatively correlated with prognosis will provide potential guiding value for the clinical research on the diagnosis and treatment of HCC.

Anti-CD47 Antibody As a Targeted Therapeutic Agent for Human Lung Cancer and Cancer Stem Cells.

Accumulating evidence indicates that a small subset of cancer cells, termed the tumor-initiating cells or cancer stem cells (CSCs), construct a reservoir of self-sustaining cancer cells with the characteristic ability to self-renew and maintain the tumor mass. The CSCs play an important role in the tumor initiation, development, relapse, metastasis, and the ineffectiveness of conventional cancer therapies. CD47 is a ligand for signal-regulatory protein-α expressed on phagocytic cells and functions to inhibit phagocytosis. This study was to explore if the expression of CD47 is the mechanism used by lung cancer cells, especially CSCs, to escape phagocytosis in vitro and in vivo. Here, we selected CD133 as the marker for lung CSCs according to previous reports. We analyzed lung cancer and matched adjacent normal (non-tumor) tissue and revealed that CD47 is overexpressed on lung cancer cells, especially on lung CSCs. The mRNA expression levels of CD47 and CD133 correlated with a decreased probability of survival for multiple types of lung cancer. Blocking CD47 function with anti-CD47 antibodies enabled macrophage phagocytosis of lung cancer cells and lung CSCs. Anti-CD47 antibodies inhibited tumor growth in immunodeficient mouse xenotransplantation models established with lung cancer cells or lung CSCs and improved survival in tumor-bearing animals. These data indicate that CD47 is a valid target for cancer therapies, especially for anti-CSC therapies.

Induction of PD-L1 expression by epidermal growth factor receptor-mediated signaling in esophageal squamous cell carcinoma.

PURPOSE: The purpose of this study was to investigate the potential effect of activation of epidermal growth factor receptor (EGFR) signaling pathway on the expression of programmed death-ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC) cells with EGFR overexpression. METHODS: Flow cytometry and Western blot methods were used to assess PD-L1 expression on ESCC cells when EGFR signaling pathway was activated by epidermal growth factor (EGF) with or without EGFR-specific inhibitor AG-1478, and then EGFR signaling array was applied to analyze the potential signaling pathways involved. RESULTS: This study found that PD-L1 expression increased significantly in an EGFR-dependent manner by the activation of EGFR signaling and decreased sharply when EGFR signaling was blocked. The upregulated expression of PD-L1 was not associated with EGFR-STAT3 signaling pathway, but may be affected by EGFR-PI3K-AKT, EGFR-Ras-Raf-Erk, and EGR-PLC-γ signaling pathways. CONCLUSION: The expression of PD-L1 can be regulated by EGFR signaling activation in ESCC, which indicates an important role for EGFR-mediated immune escape and potential molecular pathways for EGFR-targeted therapy and immunotherapy.

Programmed death-ligand 1 is prognostic factor in esophageal squamous cell carcinoma and is associated with epidermal growth factor receptor.

Programmed death-ligand 1 (PD-L1) expression either indicates immune inhibitory status or concurrent immune response. Although the relationship between PD-L1 and clinical outcomes has been studied widely in recent years, its role in prognosis of esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we assessed the significance of PD-L1 in ESCC and its association with epidermal growth factor receptor (EGFR) and radiation response. We found that PD-L1 was present both on the surface of tumor cells and tumor-infiltrating immune cells. Patients with tumor-infiltrating immune cell PD-L1 expression had better survival. PD-L1 expression on immune cells was an independent prognostic factor for patients with ESCC. PD-L1 expression either on tumor-infiltrating immune cells or tumor cells was negatively associated with EGFR expression. EGFR/PD-L1 pairs could separate the survival between EGFR low/PD-L1 positive and EGFR high/PD-L1 negative groups. In ESCC cell lines with EGFR high expression, PD-L1 expression was induced significantly when EGFR signaling was activated by radiation and was dramatically inhibited by an EGFR tyrosine kinase inhibitor. In conclusion, tumor-infiltrating immune cell PD-L1 expression is an independent prognostic factor for ESCC, and the association between EGFR and PD-L1 is vital to determining survival. It is important to consider radiotherapy-induced imbalance of pro-tumor and anti-tumor immune response. A combination of radiotherapy and PD-L1-targeted therapy could be a promising therapeutic strategy for ESCC patients.

Adoptive Cellular Therapy (ACT) for Cancer Treatment.

Adoptive cellular therapy (ACT) with various lymphocytes or antigen-presenting cells is one stone in the pillar of cancer immunotherapy, which relies on the tumor-specific T cell. The transfusion of bulk T-cell population into patients is an effective treatment for regression of cancer. In this chapter, we summarize the development of various strategies in ACT for cancer immunotherapy and discuss some of the latest progress and obstacles in technical, safety, and even regulatory aspects to translate these technologies to the clinic. ACT is becoming a potentially powerful approach to cancer treatment. Further experiments and clinical trials are needed to optimize this strategy.