A comparative study on ctDNA and tumor DNA mutations in lung cancer and benign cases with a high number of CTCs and CTECs.

BACKGROUND: Liquid biopsy provides a non-invasive approach that enables detecting circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) using blood specimens and theoretically benefits early finding primary tumor or monitoring treatment response as well as tumor recurrence. Despite many studies on these novel biomarkers, their clinical relevance remains controversial. This study aims to investigate the correlation between ctDNA, CTCs, and circulating tumor-derived endothelial cells (CTECs)  while also evaluating whether mutation profiling in ctDNA is consistent with that in tumor tissue from lung cancer patients. These findings will help the evaluation and utilization of these approaches in clinical practice. METHODS: 104 participants (49 with lung cancer and 31 with benign lesions) underwent CTCs and CTECs detection using integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy. The circulating cell-free DNA (cfDNA) concentration was measured and the mutational profiles of ctDNA were examined by Roche AVENIO ctDNA Expanded Kit (targeted total of 77 genes) by next generation sequencing (NGS) in 28 patients (20 with lung cancer and 8 with benign lesions) with highest numbers of CTCs and CTECs. Mutation validation in matched tumor tissue DNA was then performed in 9 patients with ctDNA mutations using a customized xGen pan-solid tumor kit (targeted total of 474 genes) by NGS. RESULTS: The sensitivity and specificity of total number of CTCs and CTECs for the diagnosis of NSCLC were 67.3% and 77.6% [AUC (95%CI): 0.815 (0.722-0.907)], 83.9% and 77.4% [AUC (95%CI): 0.739 (0.618-0.860)]. The concentration of cfDNA in plasma was statistically correlated with the size of the primary tumor (r = 0.430, P = 0.022) and CYFRA 21-1 (r = 0.411, P = 0.041), but not with the numbers of CTCs and CTECs. In this study, mutations were found to be poorly consistent between ctDNA and tumor DNA (tDNA) in patients, even when numerous CTCs and CTECs were present. CONCLUSION: Detection of CTCs and CTECs could be the potential adjunct tool for the early finding of lung cancer. The cfDNA levels are associated with the tumor burden, rather than the CTCs or CTECs counts. Moreover, the poorly consistent mutations between ctDNA and tDNA require further exploration.

Cancer Cells Enter an Adaptive Persistence to Survive Radiotherapy and Repopulate Tumor.

Repopulation of residual tumor cells impedes curative radiotherapy, yet the mechanism is not fully understood. It is recently appreciated that cancer cells adopt a transient persistence to survive the stress of chemo- or targeted therapy and facilitate eventual relapse. Here, it is shown that cancer cells likewise enter a "radiation-tolerant persister" (RTP) state to evade radiation pressure in vitro and in vivo. RTP cells are characterized by enlarged cell size with complex karyotype, activated type I interferon pathway and two gene patterns represented by CST3 and SNCG. RTP cells have the potential to regenerate progenies via viral budding-like division, and type I interferon-mediated antiviral signaling impaired progeny production. Depleting CST3 or SNCG does not attenuate the formation of RTP cells, but can suppress RTP cells budding with impaired tumor repopulation. Interestingly, progeny cells produced by RTP cells actively lose their aberrant chromosomal fragments and gradually recover back to a chromosomal constitution similar to their unirradiated parental cells. Collectively, this study reveals a novel mechanism of tumor repopulation, i.e., cancer cell populations employ a reversible radiation-persistence by poly- and de-polyploidization to survive radiotherapy and repopulate the tumor, providing a new therapeutic concept to improve outcome of patients receiving radiotherapy.

Comprehensive Atlas of Circulating Rare Cells Detected by SE-iFISH and Image Scanning Platform in Patients With Various Diseases.

Objective: Circulating rare cells (CRCs) are known as a crucial nucleated cellular response to pathological conditions, yet the landscape of cell types across a wide variety of diseases lacks comprehensive understanding. This study aimed at detecting and presenting a full spectrum of highly heterogeneous CRCs in clinical practice and further explored the characterization of CRC subtypes in distinct biomarker combinations and aneuploid chromosomes among various disease groups. Methods: Peripheral blood was obtained from 2,360 patients with different cancers and non-neoplastic diseases. CRC capture and identification were accomplished using a novel platform integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy with a high-throughput automated image scanning system, on which hemocyte, tumor, epithelial, endothelial, mesenchymal, and stemness biomarkers were immunostained and displayed simultaneously. Double chromosome enumeration probe (CEP8 and CEP12) co-detection was performed on isolated CRCs from an extended trial for two chromosome ploidy patterns. Results: A comprehensive atlas categorizing the diverse CRCs into 71 subtypes outlining was mapped out. The presence of epithelial-mesenchymal transition (EMT) or endothelial-mesenchymal transition (EndoMT), the cells with progenitor property, hematologic CRCs expressing multiple biomarkers, CRCs at "naked nuclei" status, and the rarely reported aneuploid mesenchymal epithelial-endothelial fusion cluster were described. Circulating tumor cells (CTCs) were detected in 2,157 (91.4%) patients; the total numbers of CTCs and circulating tumor-derived endothelial cells (CTECs) were relatively higher in several digestive system cancer types and non-neoplastic infectious diseases (p < 0.05). Co-detection combining CEP8 and CEP12 showed a higher diagnostic specificity on account of 57.27% false negativity of CRC detection through a single probe of CEP8. Conclusions: The alternative biomarkers and chromosomes to be targeted by SE-iFISH and the image scanning platform, along with the comprehensive atlas, offer insight into the heterogeneity of CRCs and reveal potential contributions to specific disease diagnosis and therapeutic target cell discovery.

Detection of circulating rare cells benefitted the diagnosis of malignant solitary pulmonary nodules.

INTRODUCTION: Solitary pulmonary nodules (SPNs) are challenging in differentiating between benignancy and malignancy. Therefore, more effective non-invasive biomarkers are urgently needed. The purpose of this investigation was to examine whether circulating rare cells (CRCs) could facilitate the differentiation between benign and malignant SPNs as well as its sensitivity and specificity. METHODS: 164 patients diagnosed with SPNs, 24 healthy volunteers, and 25 patients diagnosed with advanced-stage lung cancer were included. CT/PET-CT images, serum tumor markers, and biopsy results were collected. The CRCs were examined using subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) and their relationship with malignant or benign SPNs was analyzed. RESULTS: The total CRC numbers from patients with malignant SPNs diagnosed by biopsy were significantly greater compared to those with benign SPNs (P < 0.0001), but not significantly different from patients with advanced lung cancer (P > 0.05). The total CRCs, with a cut-off value of 21.5 units, showed 67.6% sensitivity and 73.3% specificity [area under curve (AUC) 95% CI, 0.778 (0.666-0.889)] in discriminating benign and malignant SPNs and the triploid CRCs exhibited a high positive likelihood ratio of 8.4, which suggested that CRCs appeared to have a distinct advantage in discriminating benign and malignant SPNs compared to CT/PET-CT images and serum tumor markers and could be a potential screening indicator for lung cancer in the high-risk population. CONCLUSIONS: SE-iFISH could effectively detect CRCs including circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) and the detection of CRCs could benefit the differentiation of patients with benign and malignant SPNs.

Caspase-3 knockout attenuates radiation-induced tumor repopulation via impairing the ATM/p53/Cox-2/PGE2 pathway in non-small cell lung cancer.

Radiotherapy is an effective treatment for non-small cell lung cancer (NSCLC). However, irradiated, dying tumor cells generate potent growth stimulatory signals during radiotherapy that promote the repopulation of adjacent surviving tumor cells to cause tumor recurrence. We investigated the function of caspase-3 in NSCLC repopulation after radiotherapy. We found that radiotherapy induced a DNA damage response (DDR), activated caspase-3, and promoted tumor repopulation in NSCLC cells. Unexpectedly, caspase-3 knockout attenuated the ataxia-telangiectasia mutated (ATM)/p53-initiated DDR by decreasing nuclear migration of endonuclease G (EndoG), thereby reducing the growth-promoting effect of irradiated, dying tumor cells. We also identified p53 as a regulator of the Cox-2/PGE2 axis and its involvement in caspase-3-induced tumor repopulation after radiotherapy. In addition, injection of caspase-3 knockout NSCLC cells impaired tumor growth in a nude mouse model. Our findings reveal that caspase-3 promotes tumor repopulation in NSCLC cells by activating DDR and the downstream Cox-2/PGE2 axis. Thus, caspase-3-induced ATM/p53/Cox-2/PGE2 signaling pathway could provide potential therapeutic targets to reduce NSCLC recurrence after radiotherapy.

Necroptosis regulates tumor repopulation after radiotherapy via RIP1/RIP3/MLKL/JNK/IL8 pathway.

BACKGROUND: Tumor cell repopulation after radiotherapy is a major cause for the tumor radioresistance and recurrence. This study aims to investigate the underlying mechanism of tumor repopulation after radiotherapy, with focus on whether and how necroptosis takes part in this process. METHODS: Necroptosis after irradiation were examined in vitro and in vivo. And the growth-promoting effect of necroptotic cells was investigated by chemical inhibitors or shRNA against necroptosis associated proteins and genes in in vitro and in vivo tumor repopulation models. Downstream relevance factors of necroptosis were identified by western blot and chemiluminescent immunoassays. Finally, the immunohistochemistry staining of identified necroptosis association growth stimulation factor was conducted in human colorectal tumor specimens to verify the relationship with clinical outcome. RESULTS: Radiation-induced necroptosis depended on activation of RIP1/RIP3/MLKL pathway, and the evidence in vitro and in vivo demonstrated that the inhibition of necroptosis attenuated growth-stimulating effects of irradiated tumor cells on living tumor reporter cells. The JNK/IL-8 were identified as downstream molecules of pMLKL during necroptosis, and inhibition of JNK, IL-8 or IL-8 receptor significantly reduced tumor repopulation after radiotherapy. Moreover, the high expression of IL-8 was associated with poor clinical prognosis in colorectal cancer patients. CONCLUSIONS: Necroptosis associated tumor repopulation after radiotherapy depended on activation of RIP1/RIP3/MLKL/JNK/IL-8 pathway. This novel pathway provided new insight into understanding the mechanism of tumor radioresistance and repopulation, and MLKL/JNK/IL-8 could be developed as promising targets for blocking tumor repopulation to enhance the efficacy of colorectal cancer radiotherapy.

High expression of TGR5 predicts a poor prognosis in patients with pancreatic cancer.

Previous studies have showed that bile acids (BAs) play essential roles in the progression of various human cancers, and the G-protein coupled bile acid receptor-1 (Gpbar-1, or TGR5), a receptor of BAs, has been reported to connect BAs with cancers. However, little is known about the prognostic role of TGR5 in pancreatic cancer. In this study, we found that the expression of TGR5 was significantly higher in the cancerous tissues than the adjacent normal tissues by immunohistochemical staining (81.6% vs. 36.8%). Meanwhile, TGR5 was positively correlated with lymph node metastasis (P=0.021) and advanced stage (P=0.011). Finally, univariate analysis showed that patients with high TGR5 expression (P<0.001), lymph node metastasis (P=0.002) and advanced tumor stage (P=0.008) had decreased overall survival, and Cox proportional hazards regression analysis confirmed that TGR5 expression was an independent predictor of the overall survival of patients with pancreatic cancer (P=0.019). Our findings suggested that TGR5 might serve as an important predictor of poor survival in pancreatic cancer.