Immune characteristics and genetic markers of esophageal cancer by single-cell analysis: implications for immunotherapy.

Background: Esophageal cancer (EC) is one of the most common cancers worldwide. The prognoses for patients with the same stage of EC can vary substantially. The progress of single-cell analysis technology has furthered the understanding of tumor heterogeneity. This paper aimed to apply single-cell analysis to explore the characteristics of the tumor environment of EC and provide a basis for personalized treatment. Methods: The latest gene expression data and clinical follow-up information of single-cell sequencing results of EC samples were downloaded from The Cancer Genome Atlas (TCGA) Genomic Data Commons (GDC) Application Programming Interface (API). A differential gene function analysis of the immune infiltration signature agents in the tumor microenvironment (TME) was performed using bioinformatics analytical methods, and potential molecular targets were sought. Results: We identified specific cell subsets in the EC and paracancerous samples, including panel cells, natural killer (NK) cells, exhausted cluster of differentiation (CD)8+ T cells, CD8+ memory T (Tcm) cells, and effector memory T (Tem) cells, including B cell enrichment in the cancer samples. Differences were detected between B cells and monocytes in stage II and III tumors, which may be related to RNA transcription and degradation. The CXCL8 protein was identified as a valid potential prognostic marker. Conclusions: Cell groups with homogenous cell surface markers exhibit intercellular variations that exert a considerable effect on cell function. Our study will contribute to the understanding of the TME and cellular heterogeneity in EC patients and serve as a valuable resource for in-depth exploration of the pathogenesis of EC and the identification of potential therapeutic targets in the future.

In vitro evaluation of photon and carbon ion radiotherapy in combination with cisplatin in head and neck squamous cell carcinoma cell lines.

Background: Heavy ion radiotherapy, such as carbon ion radiotherapy (CIRT), has multiple advantages over conventional photon therapy. Cisplatin, as a classic anti-tumor drugs, has been tested and discovered as a photon radiosensitizer in several cell lines, including head and neck squamous cell carcinoma (HNSCC). Hence, the aim of our study is to evaluate whether cisplatin can sensitize CIRT towards HNSCC cell lines in vitro. Methods: Human nasopharyngeal carcinoma cell line CNE-2, human tongue squamous carcinoma cell line TCA 8113 and human hypopharynx squamous carcinoma cell line FADU were all irradiated with photon beam of 2, 4, 6, 8 Gy (physical dose) and carbon ion beam of 1, 2, 3, 4 Gy (physical dose) and treated with cisplatin. Cell survival was assessed by clonogenic survival assay. Results: CIRT showed significantly stronger cytotoxic effect than standard photon radiotherapy. The relative biological effectiveness (RBE) of carbon ion beam at 10% survival ( R B E 10 ) was calculated 3.07 for CNE-2, 2.33 for TCA 8113 and 2.36 for FADU. Chemoradiotherapy (both photon radiotherapy and CIRT) was more effective than radiotherapy alone. In vitro sensitizer enhancement ratios (SERs) of cisplatin in CNE-2, TCA 8113 and FA DU cell lines after photon irradiation were 1.33, 1.14 and 1.21, while after carbon ion irradiation were 1.02, 1.00 and 0.96, showed that cisplatin sensitized photon irradiation but showed no sensitization effect in carbon ion irradiation in all tested cell lines. Conclusions: In conclusion, high linear energy transfer (LET) CIRT was more effective than photon irradiation to prevent the proliferation of HNSCC cell lines. Additional treatment with cisplatin could sensitize photon irradiation but showed no effect on carbon ion irradiation.

Treatment outcomes of 156 patients with cervical esophageal cancers treated with definitive radiation therapy- A single-institution experience of a rare cancer.

Purpose: Esophageal cancer is the most prevalent malignant tumor. The incidence of cervical esophageal cancer is low and there are insufficient data on the efficacy of radical radiotherapy. The purpose of this study was to clarify the efficacy with radical IFI radiotherapy, to analyze the pattern of initial lymph node metastasis and recurrence under the new lymph node zoning of esophageal cancer. Methods: We reviewed cervical esophageal cancer treated with radical radiotherapy. The inclusion criteria were diagnosis of esophageal cancer by pathology; receiving radical radiotherapy or chemoradiotherapy; tumor location in accordance with definition of cervical esophageal cancer. Three dimensional radiotherapy was used. The target area was IFI. Results: 156 patients entered the final analysis. The proportion of no failure was 42.31%, local esophageal failure was 30.13%, in-field lymph node metastasis was 10.26%, out-field lymph node metastasis was 1.28% and distant organ metastasis was 23.72%, second primary tumor was 2.56%. The median OS and DFS was 49.0 months (35.27-62.73) and 31.0 months (14.47-47.53). The results of initial LN metastasis pattern analysis showed the supraclavicular and upper mediastinum were the main sites of cervical esophageal cancer metastasis. In patients with recurrent LN, the results showed that the cervical, supraclavicle, upper mediastinum and abdomen were the main sites of recurrence. Conclusion: Our study is a retrospective study of a large sample of radical radiotherapy for cervical esophageal cancer. Failure in irradiation field is the main failure pattern. Concurrent radiotherapy and chemotherapy under IFI radiation is a considerable treatment option for cervical esophageal cancer.

The Impacts of Different Types of Radiation on the CRT and PDL1 Expression in Tumor Cells Under Normoxia and Hypoxia.

INTRODUCTION: Hypoxia is a hallmark of cancer that may contribute to an immunosuppressive microenvironment and promote radioresistance. High linear energy transfer (LET) radiation is considered to be able to overcome the negative effects of hypoxia. However, the anti-tumorigenic effects induced by low or high LET radiation have not been fully elucidated. This study aimed to compare the effects of different types of radiation on the immune response, particularly the impact on calreticulin (CRT), and programmed cell death ligand 1 (PDL1) expression. METHODS: Four human tumor cell lines were investigated in this study. Cells in normoxic and hypoxic groups were irradiated with 4Gy (physical dose) photon, proton, and carbon-ion radiation, respectively. The expression of CRT and PDL1 was detected 48 h after irradiation, and the median fluorescence intensities (MFIs) were compared by flow cytometry. Meanwhile, the radiosensitivity of tumor cells in each group was also compared by colony formation assays and flow cytometry. RESULTS: All types of radiation could significantly inhibit the colony formation of tumor cells under normoxia. However, the efficacy of photon and proton radiation was impaired under hypoxia. Carbon-ion radiation could still inhibit colony formation. The percentage of viable cells after irradiation was higher under hypoxia compared with those under normoxia. The CRT expression under normoxia was significantly increased after radiation. Carbon-ion radiation enhanced CRT expression compared to photon and proton radiation. Conversely, under hypoxia, the CRT expression level was significantly upregulated at baseline (0Gy). Radiation could not increase the expression further. PDL1 expression was also significantly increased by radiation under normoxia in all cell lines except the Ln18 cell line. Carbon-ion radiation induced the most significant increase. Under hypoxia, the PDL1 expression level was also upregulated at baseline and radiation could not increase expression further. CONCLUSION: Tumor cells were resistant to photon and proton but sensitive to carbon-ion radiation under hypoxia. Carbon-ion radiation could induce the highest CRT and PDL1 expression under normoxia. However, under hypoxia, radiation could not further enhance the high baseline expression of CRT and PDL1.

Depletion of MLKL inhibits invasion of radioresistant nasopharyngeal carcinoma cells by suppressing epithelial-mesenchymal transition.

BACKGROUND: To examine whether MLKL participated in the invasion of radiosensitive nasopharyngeal carcinoma (NPC) cell (CNE-2) and radioresistant NPC cell (CR) through regulating epithelial-mesenchymal transition (EMT). METHODS: siRNA and CRISPR/Cas9 technique were used to decrease MLKL expression in NPC cell (CNE-2 and CR). Trans-well assay was conducted to evaluate invasion. Gene expression profiling was performed using Human U133 2.0 plus arrays (Affymetrix). Kyoto Encyclopedia of Genes and Genomes (KEGG) was adopted to analyze gene expression profiling. Hub genes at a functional level were accessed by protein-to-protein network (PPI). Quantitative real-time PCR and Western blot were used to access EMT markers. RESULTS: Invasion of CR was about 3~fold change higher than that of CNE-2. Silencing MLKL by siRNA inhibited invasion of CR, not CNE-2. Further, depleting MLKL by CRISPR-Cas9 in CR (CR-MLKL KO) also inhibited its invasion. KEGG pathway analysis showed invasion-related pathways were altered, such as adherent junction, TGF-ß signaling pathway. PPI demonstrated that compared with CNE-2, CR showed 9 elevated hub genes including EGFR, JUN, CD44, SPP1, VIM, IL-8, BCL2, WDFY2, PIK3CD and 1 downregulated hub gene CDH1. After MLKL depletion, 8 hub genes were downregulated (EGFR, JUN, CD44, SPP1, VIM, FGF13, PLAU, MMP1) and 2 hub genes were upregulated (MMP9, CDH1). Quantitative real-time PCR results showed that compared with CNE-2, CR displayed decreased epithelial markers significantly (E-Cadherin) and increased mesenchymal markers significantly (Vimentin, N-Cadherin, Zeb1), indicating irradiation-induced EMT. After depletion of MLKL in CR, the expression of E-Cadherin, Vimentin, N-Cadherin, Zeb1 was reversed to the level of CNE-2. Western blot confirmed the results from qRT-PCR. CONCLUSIONS: Depletion of MLKL efficiently inhibits invasion of radioresistant NPC by suppressing EMT. MLKL may be an important target to suppress distant metastasis of NPC patients who relapsed after radiotherapy.