Chemosensitivity Of Tumor Cells Research Articles

Knocking down RAD51AP1 enhances chemosensitivity by inhibiting the self-renewal of CD133 positive ovarian cancer stem-like cells

PurposeThis study was designed to investigate the function of RAD51AP1 in the self-renewal and chemosensitivity of CD133 positive (CD133+) ovarian cancer (OC) stem-like cells.MethodsCD133+ (CD133 positive) OVCAR4 and CD133 negative (CD133−) OVCAR4 cells were separated from OVCAR4 by flow cytometry. Then, the separated CD133+OVCAR4 cells were divided into the following groups: Vector group; RAD51AP1 group; siNC group; si-RAD51AP1 group. Next, sphere-formation assay and colony forming assay were used to evaluate the self-renewal and proliferation ability of cells; western blot to detect the expression of RAD51AP1, transforming growth factor beta 1 (TGF-β1) and SMAD4 proteins in tissues and cells; qRT-PCR to assess the mRNA levels of sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), NANOG and Kruppel-like factor 4 (KLF4).ResultsThe performance of CD133+OVCAR4 cells was much better than that of CD133−OVCAR4 cells in sphere-formation assay and colony forming assay. Besides, compared with adjacent group and CD133−OVCAR4 cells, the expression level of RAD51AP1 increased significantly in OC group and CD133+OVCAR4 cells. Moreover, the over-expression of RAD51AP1 promoted the self-renewal and proliferation of CD133+OVCAR4 cells. On the contrary, knocking down the expression level of RAD51AP1 could inhibit the self-renewal and proliferation of CD133+OVCAR4 cells and improve the sensitivity of cells to chemotherapy drugs.ConclusionThe findings of this study showed that RAD51AP1 was highly expressed in OC tissue and CD133+OVCAR4 cells, and regulated the self-renewal and chemosensitivity of tumor cells through the TGF-β1/SMAD4 signaling pathway.

The small phytomolecule resveratrol: A promising role in boosting tumor cell chemosensitivity

Resveratrol (RES), chemically known as trans-3,5,4′-trihydroxystilbene, is a polyphenolic molecule that occurs naturally and is produced by a variety of plants in response to being stimulated by diverse stimuli. It possesses a wide range of biological activities and provides a multitude of health benefits, including anti-tumor, cardioprotective, anti-inflammatory, and antioxidant characteristics. According to the findings of research on the bioavailability of RES, oral administration results in a high level of absorption. However, research has demonstrated that the administration of RES through gavage or intravenous administration produces more favorable results than the administration of RES through oral administration. As a result, more research has been carried out to address the rapid metabolism of RES. This has been accomplished through the utilization of novel formulation methodologies, metabolic regulation, and the analysis of potential interactions with other dietary variables. Through the process of triggering apoptosis, RES has been proposed as a possible agent for reversing drug resistance and improving the therapeutic potential of chemotherapy. Additionally, RES exhibits promising antiproliferative properties when paired with chemotherapeutic medicines, which enhances the overall function of these treatments. It is vital to do additional research to shed light on the beneficial role that RES plays in the context of cancer therapy, even though there have been few clinical trials that combine RES with anticancer medications.

Cyclic Sesquiterpene-Flavanone [4+2] Hybrids, Syzygioblanes A-C, Found in an Indonesian Traditional Medicine, "Jampu Salo" (Syzygium oblanceolatum).

A plant used in an Indonesian traditional herbal medicine as a diabetes treatment and known locally as "Jampu Salo" was collected on Sulawesi Island, Indonesia. It was identified as Syzygium oblanceolatum (C. B. Rob.) Merr. (Myrtaceae) and found for the first time in Sulawesi; it was previously reported only in the eastern Philippines and Borneo. A phytochemical study of S. oblanceolatum led to the isolation of three unprecedented meroterpenoids, syzygioblanes A-C (1-3, respectively). These compounds might be biosynthesized through [4+2] cycloaddition of various germacrane-based cyclic sesquiterpenoids with the flavone desmethoxymatteucinol to form a spiro skeleton. The unique and complex structures were elucidated by microcrystal electron diffraction analysis in addition to general analytical techniques such as high-resolution mass spectrometry, various nuclear magnetic resonance methods, and infrared spectroscopy. Synchrotron X-ray diffraction and calculations of electronic circular dichroism spectra helped to determine the absolute configurations. The newly isolated compounds exhibited collateral sensitivity to more strongly inhibit the growth of a multidrug resistant tumor cell line compared to a chemosensitive tumor cell line.

Understanding the Role of Connexins in Hepatocellular Carcinoma: Molecular and Prognostic Implications.

Connexins, a family of tetraspan membrane proteins forming intercellular channels localized in gap junctions, play a pivotal role at the different stages of tumor progression presenting both pro- and anti-tumorigenic effects. Considering the potential role of connexins as tumor suppressors through multiple channel-independent mechanisms, their loss of expression may be associated with tumorigenic activity, while it is hypothesized that connexins favor the clonal expansion of tumor cells and promote cell migration, invasion, and proliferation, affecting metastasis and chemoresistance in some cases. Hepatocellular carcinoma (HCC), characterized by unfavorable prognosis and limited responsiveness to current therapeutic strategies, has been linked to gap junction proteins as tumorigenic factors with prognostic value. Notably, several members of connexins have emerged as promising markers for assessing the progression and aggressiveness of HCC, as well as the chemosensitivity and radiosensitivity of hepatocellular tumor cells. Our review sheds light on the multifaceted role of connexins in HCC pathogenesis, offering valuable insights on recent advances in determining their prognostic and therapeutic potential.

Exploring the mechanism of contact-dependent cell-cell communication on chemosensitivity based on single-cell high-throughput drug screening platform

High-throughput drug screening (HTDS) has significantly reduced the time and cost of new drug development. Nonetheless, contact-dependent cell-cell communication (CDCCC) may impact the chemosensitivity of tumour cells. There is a pressing need for low-cost single-cell HTDS platforms, alongside a deep comprehension of the mechanisms by which CDCCC affects drug efficacy, to fully unveil the efficacy of anticancer drugs. In this study, we develop a microfluidic chip for single-cell HTDS and evaluate the molecular mechanisms impacted by CDCCC using quantitative mass spectrometry-based proteomics. The chip achieves high-quality drug mixing and single-cell capture, with single-cell drug screening results on the chip showing consistency with those on the 96-well plates under varying concentration gradients. Through quantitative proteomic analysis, we deduce that the absence of CDCCC in single tumour cells can enhance their chemoresistance potential, but simultaneously subject them to stronger proliferation inhibition. Additionally, pathway enrichment analysis suggests that CDCCC could impact several signalling pathways in tumour single cells that regulate vital biological processes such as tumour proliferation, adhesion, and invasion. These results offer valuable insights into the potential connection between CDCCC and the chemosensitivity of tumour cells. This research paves the way for the development of single-cell HTDC platforms and holds the promise of advancing tumour personalized treatment strategies.

PPARG and the PTEN-PI3K/AKT Signaling Axis May Cofunction in Promoting Chemosensitivity in Hypopharyngeal Squamous Cell Carcinoma.

It has been demonstrated that PPARG may interact with the PTEN-PI3K/AKT pathway, contributing to its involvement in the chemotherapy treatment of hypopharyngeal squamous cell carcinoma (HSCC). However, the underlying mechanism remains largely unknown. In this study, gene expression profiles of 17 HSCC patients, comprising 8 chemotherapy-sensitive patients (CSP) and 9 chemotherapy-nonsensitive patients (CNSP), were collected and analyzed to investigate expression patterns, correlations, influencing factors of the PPARG-PTEN-PI3K/AKT pathway, and its role in regulating chemosensitivity. The results revealed significantly increased expression (p < 0.04) of AKT1, AKT2, AKT3, PIK3CA, PPARG, and PTEN in the CSP group compared to the CNSP group. Specifically, AKT2 exhibited significant overexpression in tumor tissue (p = 0.01), while AKT2, AKT3, PPARG, and PTEN displayed significant increases in normal tissue (p ≤ 0.04). Positive correlations (R ∈ [0.43, 0.71], p < 0.014) were observed between PIK3CA, AKT1, AKT2, AKT3, and PTEN, with AKT2, AKT3, and PTEN also showing significant correlations with PPARG (R ∈ [0.35, 0.47], p < 0.04). Age, gender, and disease stage had no influence on PPARG, PIK3CA, and PTEN expression, but they may affect AKT expressions. Pathway analysis revealed that PPARG may interact with the PTEN-PI3K/AKT signaling pathway, playing a crucial role in regulating chemosensitivity in the normal tissue microenvironment. Our results suggest that AKT1 and PIK3CA may be associated with chemosensitivity in HSCC tumor cells, while PPARG and PTEN might exhibit a correlation with a specific segment of the PI3K/AKT pathway, potentially influencing chemosensitivity in the normal tissue microenvironment of HSCC patients.

ONCOBREAST-TEST Is a Quick Diagnostic, Prognostic and Predictive Method of Response to Systemic Treatment.

ONCOBREAST-TEST is a diagnostic and therapeutic procedure that is part of the comprehensive care of a patient with breast cancer.: Chemosensitivity of cancer cells was assessed using the MTT test, morphological assessment of cells, LDH activity in the culture medium, and flow cytometry technique (apoptosis, proliferation, CD24, CD44, GATA3, cytokeratin, Ki-67). Diagnostic tools included panels of simple tests which could be used to accurately predict the chemosensitivity of tumor cells previously isolated from a patient, even before actual chemotherapy. The proposed procedure allows for a simple (based on MTT results, cell morphology, LDH concentration), minimally invasive, quick, and accurate assessment of the sensitivity of breast cancer cells to the drugs used and, to select the most effective treatment plan as part of personalized therapy. In a patient with NOS G3, the most promising therapy will be docetaxel with cyclophosphamide and in the case of a patient with NOS G1, paclitaxel alone and in combination with trastuzumab. The implementation of such a procedure would undoubtedly increase the effectiveness of chemotherapy, reduce side effects by excluding drugs that are ineffective before using them, protect the patient's health, and shorten the treatment time, bringing economic and social benefits.

Molecular mechanisms of action and chemosensitization of tumor cells in ovarian cancer by phytochemicals: A narrative review on pre-clinical and clinical studies.

Ovarian cancer is the second-leading cause of death among women with cancer of the genital tract. Currently, drugs derived from platinum and taxanes constitute the majority of ovarian cancer treatments. Patients undergoing this chemotherapy are susceptible to cumulative toxic effects and resistance to chemotherapy. Therefore, it is crucial to identify treatment options that are both more effective and better tolerated by patients. Phytochemicals in this context are plant-derived chemicals with antitumor activity that can be used as therapeutic or adjuvant agents in the treatment of ovarian cancer. Consequently, the purpose of this literature review is to demonstrate through existing pre-clinical and clinical trials the potential of phytochemicals in the treatment of ovarian cancer, the mechanisms of action involved, and to contribute to the development of new therapeutic options for ovarian cancer. For this review, the databases PubMed, Scopus, Science Direct, and ClinicalTrials.gov were queried between 2010 and 2022 using terms such as "ovarian cancer," "phytochemicals," "phenolic compounds," "terpenes," and "alkaloids." The present review summarized the possible molecular mechanisms of action by which phytochemicals, such as phenolic acids, flavonoids, diterpenes, triterpenes, saponins, and alkaloids, inhibit this type of cancer, specifically the ability of phytochemicals to induce cell growth regulation, apoptosis, oxidative stress reduction, anti-angiogenesis, and chemosensitization of tumors in ovarian cancer. As their action and cellular mechanism have already been demonstrated in several pre-clinical trials, the phytochemicals identified in our study have the potential to be investigated for the treatment of ovarian cancer. Through pre-clinical and clinical trials, our study demonstrates the potential of phytochemicals in the treatment of ovarian cancer, contributing to the development of novel therapeutic options for ovarian cancer.

Targeted downregulation of MYC mediated by a highly efficient lactobionic acid-based glycoplex to enhance chemosensitivity in human hepatocellular carcinoma cells

The chemosensitization of tumor cells by gene therapy represents a promising strategy for hepatocellular carcinoma (HCC) treatment. In this regard, HCC-specific and highly efficient gene delivery nanocarriers are urgently needed. For this purpose, novel lactobionic acid-based gene delivery nanosystems were developed to downregulate c-MYC expression and sensitize tumor cells to low concentration of sorafenib (SF). A library of tailor-made cationic glycopolymers, based on poly(2-aminoethyl methacrylate hydrochloride) (PAMA) and poly(2-lactobionamidoethyl methacrylate) (PLAMA) were synthesized by a straightforward activators regenerated by electron transfer atom transfer radical polymerization. The nanocarriers prepared with PAMA114-co-PLAMA20 glycopolymer were the most efficient for gene delivery. These glycoplexes specifically bound to the asialoglycoprotein receptor and were internalized through the clathrin-coated pit endocytic pathway. c-MYC expression was significantly downregulated by MYC short-hairpin RNA (MYC shRNA), resulting in efficient inhibition of tumor cells proliferation and a high levels apoptosis in 2D and 3D HCC-tumor models. Moreover, c-MYC silencing increased the sensitivity of HCC cells to SF (IC50 for MYC shRNA + SF 1.9 μM compared to 6.9 μM for control shRNA + SF). Overall, the data obtained demonstrated the great potential of PAMA114-co-PLAMA20/MYC shRNA nanosystems combined with low doses of SF for the treatment of HCC.

Fermented Ginger Extract in Natural Deep Eutectic Solvent Enhances Cytotoxicity by Inhibiting NF-κB Mediated CXC Chemokine Receptor 4 Expression in Oxaliplatin-Resistant Human Colorectal Cancer Cells.

Ginger extracts have been shown to have health-promoting pharmacological activity and beneficial effects, including antioxidant and anticancer properties. The extraction of ginger by natural deep eutectic solvents (NaDES) has been shown to enhance bioactivity, but the cytotoxicity of NaDES extracts needs to be further determined. Signaling through the CXC chemokine receptor 4 (CXCR4) expressed on colorectal cancer (CRC) cells has a pivotal role in tumor cell chemosensitivity. Oxaliplatin is a third-generation platinum compound used as an effective chemotherapeutic drug for CRC treatment. However, whether ginger extract and oxaliplatin could induce a synergistic cytotoxic effect in oxaliplatin-resistant CRC cells through modulating CXCR4 expression is not known. In this study, oxaliplatin-resistant HCT-116 (HCT-116/R) cells were generated first. Ginger was extracted using the NaDES mixture betaine/lactate/water (1:2:2.5). Lactobacillus reuteri fermentation of NaDES-ginger extract increased the total polyphenol content (12.42 mg gallic acid/g in non-fermented NaDES-ginger extract and 23.66 mg gallic acid/g in fermented NaDES-ginger extract). It also increased the antioxidant activity by about 20–30% compared to non-fermented NaDES-ginger extract. In addition, it achieved low cytotoxicity to normal colonic mucosal cells and enhanced the anticancer effect on HCT-116/R cells. On the other hand, the inhibition of NF-κB activation by fermented NaDES-ginger extract significantly decreased the CXCR4 expression (p < 0.05) in HCT-116/R cells. The inactivation of NF-κB by pharmacological inhibitor pyrrolidine dithiocarbamate further enhanced the fermented NaDES-ginger extract-reduced CXCR4 expression levels (p < 0.05). Moreover, fermented NaDES-ginger extract could synergistically increase the cytotoxicity of oxaliplatin by inhibiting CXCR4 expression and inactivating NF-κB, resulting in HCT-116/R cell death. These findings demonstrate that fermented NaDES-ginger extract reduces the NF-kB-mediated activation of CXCR4 and enhances oxaliplatin-induced cytotoxicity in oxaliplatin-resistant CRC cells.

A 3D Collagen-Based Bioprinted Model to Study Osteosarcoma Invasiveness and Drug Response.

The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.

Glutor, a Glucose Transporter Inhibitor, Exerts Antineoplastic Action on Tumor Cells of Thymic Origin: Implication of Modulated Metabolism, Survival, Oxidative Stress, Mitochondrial Membrane Potential, pH Homeostasis, and Chemosensitivity.

Neoplastic cells overexpress glucose transporters (GLUT), particularly GLUT1 and GLUT3, to support altered metabolism. Hence, novel strategies are being explored to effectively inhibit GLUTs for a daunting interference of glucose uptake. Glutor, a piperazine-2-one derivative, is a newly reported pan-GLUT inhibitor with a promising antineoplastic potential. However, several aspects of the underlying mechanisms remain obscure. To understand this better, tumor cells of thymic origin designated as Dalton’s lymphoma (DL) were treated with glutor and analyzed for survival and metabolism regulatory molecular events. Treatment of tumor cells with glutor caused a decrease in cell survival with augmented induction of apoptosis. It also caused a decrease in glucose uptake associated with altered expression of GLUT1 and GLUT3. HIF-1α, HK-2, LDH-A, and MCT1 also decreased with diminished lactate production and deregulated pH homeostasis. Moreover, glutor treatment modulated the expression of cell survival regulatory molecules p53, Hsp70, IL-2 receptor CD25, and C-myc along with mitochondrial membrane depolarization, increased intracellular ROS expression, and altered Bcl-2/BAX ratio. Glutor also enhanced the chemosensitivity of tumor cells to cisplatin, accompanied by decreased MDR1 expression. Adding fructose to the culture medium containing glutor reversed the latter’s inhibitory action on tumor cell survival. These results demonstrate that in addition to inhibited glucose uptake, modulated tumor growth regulatory molecular pathways are also implicated in the manifestation of the antineoplastic action of glutor. Thus, the novel findings of this study will have a long-lasting clinical significance in evaluating and optimizing the use of glutor in anticancer therapeutic strategies.

Circulating tumour cell gene expression and chemosensitivity analyses: predictive accuracy for response to multidisciplinary treatment of patients with unresectable refractory recurrent rectal cancer or unresectable refractory colorectal cancer liver metastases

BackgroundPatients with unresectable recurrent rectal cancer (RRC) or colorectal cancer (CRC) with liver metastases, refractory to at least two lines of traditional systemic therapy, may receive third line intraarterial chemotherapy (IC) and targeted therapy (TT) using drugs selected by chemosensitivity and tumor gene expression analyses of liquid biopsy-derived circulating tumor cells (CTCs).MethodsIn this retrospective study, 36 patients with refractory unresectable RRC or refractory unresectable CRC liver metastases were submitted for IC and TT with agents selected by precision oncotherapy chemosensitivity assays performed on liquid biopsy-derived CTCs, transiently cultured in vitro, and by tumor gene expression in the same CTC population, as a ratio to tumor gene expression in peripheral mononuclear blood cells (PMBCs) from the same individual. The endpoint was to evaluate the predictive accuracy of a specific liquid biopsy precision oncotherapy CTC purification and in vitro culture methodology for a positive RECIST 1.1 response to the therapy selected.ResultsOur analyses resulted in evaluations of 94.12% (95% CI 0.71–0.99) for sensitivity, 5.26% (95% CI 0.01–0.26) for specificity, a predictive value of 47.06% (95% CI 0.29–0.65) for a positive response, a predictive value of 50% (95% CI 0.01–0.98) for a negative response, with an overall calculated predictive accuracy of 47.22% (95% CI 0.30–0.64).ConclusionsThis is the first reported estimation of predictive accuracy derived from combining chemosensitivity and tumor gene expression analyses on liquid biopsy-derived CTCs, transiently cultured in vitro which, despite limitations, represents a baseline and benchmark which we envisage will be improve upon by methodological and technological advances and future clinical trials.

Fanconi Anemia Pathway in Colorectal Cancer: A Novel Opportunity for Diagnosis, Prognosis and Therapy.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and has the second highest mortality rate globally. Thanks to the advent of next-generation sequencing technologies, several novel candidate genes have been proposed for CRC susceptibility. Germline biallelic mutations in one or more of the 22 currently recognized Fanconi anemia (FA) genes have been associated with Fanconi anemia disease, while germline monoallelic mutations, somatic mutations, or the promoter hypermethylation of some FANC genes increases the risk of cancer development, including CRC. The FA pathway is a substantial part of the DNA damage response system that participates in the repair of DNA inter-strand crosslinks through homologous recombination (HR) and protects genome stability via replication fork stabilization, respectively. Recent studies revealed associations between FA gene/protein tumor expression levels (i.e., FANC genes) and CRC progression and drug resistance. Moreover, the FA pathway represents a potential target in the CRC treatment. In fact, FANC gene characteristics may contribute to chemosensitize tumor cells to DNA crosslinking agents such as oxaliplatin and cisplatin besides exploiting the synthetic lethal approach for selective targeting of tumor cells. Hence, this review summarizes the current knowledge on the function of the FA pathway in DNA repair and genomic integrity with a focus on the FANC genes as potential predisposition factors to CRC. We then introduce recent literature that highlights the importance of FANC genes in CRC as promising prognostic and predictive biomarkers for disease management and treatment. Finally, we represent a brief overview of the current knowledge around the FANC genes as synthetic lethal therapeutic targets for precision cancer medicine.

Persistent EGFR/K-RAS/SIAH pathway activation drives chemo-resistance and early tumor relapse in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It disproportionately affects BRCA mutation carriers and young women, especially African American (AA) women. Chemoresistant TNBC is a heterogeneous and molecularly unstable disease that challenges our ability to apply personalized therapies. With the approval of immune checkpoint blockade (ICB) for TNBC, the addition of pembrolizumab to systemic chemotherapy has become standard of care (SOC) in neoadjuvant systemic therapy (NST) for high-risk early-stage TNBC. Pembrolizumab plus chemotherapy significantly increased the pathologic complete response (pCR) and improved event-free survival in TNBC. However, clinical uncertainties remain because similarly treated TNBC partial responders with comparable tumor responses to neoadjuvant therapy often experience disparate clinical outcomes. Current methods fall short in accurately predicting which high-risk patients will develop chemo-resistance and tumor relapse. Therefore, novel treatment strategies and innovative new research initiatives are needed. We propose that the EGFR-K-RAS-SIAH pathway activation is a major tumor driver in chemoresistant TNBC. Persistent high expression of SIAH in residual tumors following NACT/NST reflects that the EGFR/K-RAS pathway remains activated (ON), indicating an ineffective response to treatment. These chemoresistant tumor clones persist in expressing SIAH (SIAHHigh/ON) and are linked to early tumor relapse and poorer prognosis. Conversely, the loss of SIAH expression (SIAHLow/OFF) in residual tumors post-NACT/NST reflects EGFR/K-RAS pathway inactivation (OFF), indicating effective therapy and chemo-sensitive tumor cells. SIAHLow/OFF signal is linked to tumor remission and better prognosis post-NACT/NST. Therefore, SIAH is well-positioned to become a novel tumor-specific, therapy-responsive, and prognostic biomarker. Potentially, this new biomarker (SIAHHigh/ON) could be used to quantify therapy response, predict chemo-resistance, and identify those patients at the highest risk for tumor relapse and poor survival in TNBC.

Chemosensitivity Evaluation using Circulating Tumor Cells Predicts Chemotherapy Outcomes in Patients with Advanced Cancer.

Chemotherapy is a clinically recognized effective technique for systemic treatment of malignant tumors. However, the tumor heterogeneity and multiple drug resistance (MDR) to the chemotherapeutic agents often lead to a failure of response to chemotherapy. We utilized a novel in vitro chemosensitivity test to identify sensitive and effective chemotherapeutic drugs and further elucidated the correlation between the in vitro chemosensitivity and clinical outcomes. We developed a circulating tumor cell-based in vitro drug sensitivity test to evaluate the sensitivity of different chemotherapeutic agents. High glucose uptake combined with negative CD45 marker were exploited to distinguish the CTCs from leukocytes. The altered glucose metabolism of single cell was measured by custom-designed computational algorithm, and the toxicity of different drug combinations was assessed by different fluorescent intensity on CTCs in the treated and control group. We analyzed the potential of CTCs in predicting chemotherapy response in 92 patients with different cancer types. Our data showed that the isolated CTCs accurately predicted chemotherapy outcomes, especially in patients with late-stage cancer. CTC-based chemosensitivity evaluation can help guide clinical decision making and identify patients who are likely to benefit from chemotherapy. CTC-based chemosensitivity evaluation is an effective methodology to study the chemosensitivity of tumor cells in vitro. Our results using CTC-based chemosensitivity evaluation method were well correlated with the clinical outcomes of chemotherapy. The clinical implementation of our CTC-based chemosensitivity evaluation method can help spare patients with primary chemoresistance from the unnecessary toxicities of chemotherapy and improve chemotherapy outcomes.

Synergism between IGF2BP1 and ETV6-RUNX1 in the Pathogenesis of ETV6-RUNX1 Positive B-Acute Lymphoblastic Leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common pediatric haematological malignancy with a prevalence of around 85% cases of B cell origin. ETV6-RUNX1 is the most common genetic aberration in childhood B-ALL with a prevalence of around 25% in Western countries and a lower prevalence in Asian countries. ETV6-RUNX1 presence portends a good prognosis in B-ALL. ETV6-RUNX1 translocation has been observed in 1-5% of healthy newborns, indicating that it is not sufficient for leukemogenesis, and the need to accumulate secondary mutations for leukemogenesis.We have previously demonstrated the specific overexpression of Insulin like Growth Factor 2 Binding Protein 1 (IGF2BP1), an oncofetal RNA binding protein, in ETV6-RUNX1 positive B-ALL. Overexpression of the ETV6-RUNX1 fusion protein in 7OZ/3, a murine B-ALL cell line increased endogenous Igf2bp1 levels in a dose dependent manner.Conversely, knockout of IGF2BP1 in Reh-Cas9-GFP expressing B-ALL cell line, a modified version of Reh (cell line positive for ETV6-RUNX1 translocation), using 2 different guide RNAs led to a decrease in the ETV6-RUNX1 transcript expression in the IGF2BP1 KO Reh-Cas9 cells. This was accompanied by a decrease in cell proliferation and increased chemosensitivity to prednisolone.A similar phenotype was observed in Reh-Cas9 cells after knockout of ETV6-RUNX1 fusion transcript hinting towards a synergism between the two proteins in tumour cell survival and chemosensitivity. Interestingly, there appears to be a positive correlation between prednisolone resistance and ETV6-RUNX1 positivity in our patient cohort too, thus supporting the in vitro finding. Previous studies have shown the binding of IGF2BP1 to the ETV6-RUNX1 fusion transcript which was also confirmed by us.To study the in vivo synergistic effect between ETV6-RUNX1 fusion protein and IGF2BP1, we used a murine bone marrow transplant model. The individual genes were overexpressed alone (IGF2BP1/ETV6-RUNX1) or together (IGF2BP1+ETV6-RUNX1) in separate groups of lethally irradiated mice (n=8 per group). In the group where both the genes were overexpressed together, there was clonal expansion and hypercellularity in the bone marrow. This was accompanied by an increase in the number of lineage negative cells (Lin-), progenitors including HSCs, LMPPs and c-kit+ cells which also showed high Ki67 positivity. The marrow hypercellularity led to extramedullary haematopoiesis which was seen as significant splenomegaly.In addition, analysis of the endogenous levels of Igf2bp1 in the bone marrow of the primary murine samples belonging to the ETV6-RUNX1 fusion protein overexpression group resulted in an increase in its levels, thus confirming our in vitro finding suggesting its induction by ETV6-RUNX1 fusion protein.RNA-Seq of IGF2BP1 KO cells identified enrichment of targets of the TNF alpha/NFκB signalling and K-Ras pathways hinting towards their dependency on IGF2BP1. The direct targets of IGF2BP1 were identified by RIP-Seq (RNA-Immunoprecipitation-Seq) analysis in Reh cell line. Numerous genes from the oncogenic signalling pathways including the TNF alpha/ NFκB signalling pathway were identified as IGF2BP1 targets. We validated some overexpressed genes from the TNF alpha/NFκB signalling and PI3K pathway in primary patient samples. We found significant overexpression of some of the genes (IL6ST, NFAT5, CDK6, MDM2, CCND1, NGFR) belonging to these pathways in the ETV6-RUNX1 positive group (n=39) compared to the negative cohort (n=111) as well as MACS sorted CD19+ B-cells (n=5). The decrease in the ETV6-RUNX1 transcript levels after downregulation of IGF2BP1 and overlap between the oncogenic pathways clearly shows an interdependency between the two genes.Overall, our results suggest a potential feedback mechanism between ETV6-RUNX1 fusion protein and IGF2BP1 in the pathogenesis of leukemia development and IGF2BP1 can be utilised as an ideal therapeutic target for this particular subtype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Microribonucleic Acid-15a-5p Alters Adriamycin Resistance in Breast Cancer Cells by Targeting Cell Division Cycle-Associated Protein 4.

ObjectiveAlthough chemotherapy is one of the first line clinical treatment of tumors, the efficacy of chemotherapy has been severely restricted by the frequent occurrence of drug resistance phenomenon. Multiple studies found that miRNAs can regulate the chemosensitivity of tumor cells. Here, this study aimed to assess the potential role of the miR-15a-5p/cell division cycle-related protein 4 (CDCA4) axis in breast cancer (BC) resistance to Adriamycin.MethodsIn the present study, the relative expression of miRNA-15a-5p in MCF-7/ADR, MCF-7 and Hs578Bst was measured by qRT-PCR. MCF-7/ADR cells underwent transfection with an miR-15a-5p mimic and inhibitor, respectively. Transwell assays, flow cytometry and CCK8 were performed to examine the potential effects of the abnormal expression of miR-15a-5p. The association of aberrant miR-15a-5p expression with Adriamycin resistance in BC was determined in cultured MCF-7/ADR cells. Bioinformatics was employed to predict the genes targeted by miR-15a-5p. Moreover, the correlation between miR-15a-5p and its target gene, CDCA4, was evaluated based on qRT-PCR data.ResultsThe expression of miR-15a-5p was significantly downregulated in MCF/ADR cells compared with MCF-7 and Hs578Bst cell lines. In the presence of Adriamycin, miR-15a-5p overexpression significantly increased cell chemosensitivity, as well as MCF-7/ADR cell proliferation, invasion, and migration, while promoting apoptosis and inducing cell-cycle arrest in the synthesis phase. CDCA4 RNA interference enhanced these effects as shown in our previous study. Bioinformatics identified CDCA4 as an miR-15a-5p target gene. qRT-PCR further demonstrated that CDCA4 and miR-15a-5p expression levels were inversely correlated.ConclusionAdriamycin resistance in BC cells was, at least in part, altered by mRNA-15a-5p via regulation of its target gene, CDCA4, by controlling the cell cycle, which may provide some novel ideas for BC chemotherapy in the future.

Comparison of the Potency of Clinically Relevant Platinum Derivatives to Enhance the Abscopal Effect

Localized radiotherapy (RT) can cause T-cell-mediated abscopal effects on non-irradiated metastases, particularly in combination with immune checkpoint blockade (ICB). However, results of prospective clinical trials have not met the expectations. We study whether chemotherapeutics can enhance the abscopal effect. Oxaliplatin (Oxa) has been considered as immunogenic, but cisplatin (Cis) and carboplatin (Carbo) not. We compared these three platinum derivatives in two different abscopal mouse models.In mice bearing bilateral tumors, the primary tumor was irradiated with 2 × 12 Gy (B16 melanoma model) or 2 × 8 Gy (C51 colon carcinoma model); Cis, Carbo, or Oxa were given once together with RT; anti-PD1 was given weekly. Tumor growth and survival of mice were determined (5-15 mice per group). Dependence of therapeutic effects on CD8+ T cells and extracellular ATP were determined by using T-cell-depleting antibodies and PPADS (a nontoxic broad-spectrum inhibitor of purinergic receptors), respectively. Frequencies and functionality (differentiation and exhaustion state) of tumor-specific CD8+ T cells were determined by FACS using MHC tetramers and various antibodies. In vitro, we determined the chemosensitivity of the tumor cell lines and their production of extracellular ATP.The enhancement of the abscopal effect compared to RT/αPD-1 was as follows: RT/αPD-1/Cis (P < 0.01) > RT/αPD-1/Oxa (P < 0.01) > > RT/αPD-1/Carbo (P > 0.05) (B16 melanoma); RT/αPD-1/Cis (P < 0.001) ≈ RT/αPD-1/Carbo (P < 0.01) > > RT/αPD-1/Oxa (P > 0.05) (C51 model). Triple therapy with Cis resulted in complete abscopal regression in 7/15 and 8/9 mice bearing B16 or C51 tumors, respectively. Of note, triple therapy with Cis was also significantly better than the Cis/αPD-1 double combination in both tumor models (B16 model, P < 0.01; C51 model, P < 0.05). In vitro, the chemosensitivity was as follows: Cis ≈ Oxa > Carbo (B16 melanoma); Cis ≈ Carbo > Oxa (C51 colon carcinoma). Cis and Carbo induced more extracellular ATP than Oxa in both tumor cell lines. In the C51 model, triple therapy with Cis induced more mature dendritic cells (P < 0.05), more Ki67+ CD8+ T cells (P < 0.05), and more tumor-specific T cells (P < 0.01) compared to triple therapy with Oxa. The enhanced abscopal effect was abrogated when CD8+ T cells were depleted or extracellular ATP signaling was blocked.Both the chemosensitivity of tumor cells and the potency of chemotherapeutics to induce ATP secretion may be crucial for the platinum-mediated enhancement of the abscopal effect. Our findings may be important for the planning of clinical trials of triple combinations of RT, chemotherapy, and ICB in metastatic patients.

Experimental and Modelling Analysis of the Hyperthermia Properties of Iron Oxide Nanocubes.

The ability of magnetic nanoparticles (MNPs) to transform electromagnetic energy into heat is widely exploited in well-known thermal cancer therapies, such as magnetic hyperthermia, which proves useful in enhancing the radio- and chemo-sensitivity of human tumor cells. Since the heat release is ruled by the complex magnetic behavior of MNPs, a careful investigation is needed to understand the role of their intrinsic (composition, size and shape) and collective (aggregation state) properties. Here, the influence of geometrical parameters and aggregation on the specific loss power (SLP) is analyzed through in-depth structural, morphological, magnetic and thermometric characterizations supported by micromagnetic and heat transfer simulations. To this aim, different samples of cubic Fe3O4 NPs with an average size between 15 nm and 160 nm are prepared via hydrothermal route. For the analyzed samples, the magnetic behavior and heating properties result to be basically determined by the magnetic single- or multi-domain configuration and by the competition between magnetocrystalline and shape anisotropies. This is clarified by micromagnetic simulations, which enable us to also elucidate the role of magnetostatic interactions associated with locally strong aggregation.