A highly integrated digital PCR system with on-chip heating for accurate DNA quantitative analysis.

Digital polymerase chain reaction (dPCR) is extensively used for highly sensitive disease diagnosis due to its single-molecule detection ability. However, current dPCR systems require intricate DNA sample distribution, rely on cumbersome external heaters, and exhibit sluggish thermal cycling, hampering efficiency and speed of the dPCR process. Herein, we presented the development of a microwell array based dPCR system featuring an integrated self-heating dPCR chip. By utilizing hydrodynamic and electrothermal simulations, the chip's structure is optimized, resulting in improved partitioning within microwells and uniform thermal distribution. Through strategic hydrophilic/hydrophobic modifications on the chip's surface, we effectively secured the compartmentalization of sample within the microwells by employing an overlaying oil phase, which renders homogeneity and independence of samples in the microwells. To achieve precise, stable, uniform, and rapid self-heating of the chip, the ITO heating layer and the temperature control algorithm are deliberately designed. With a capacity of 22,500 microwells that can be easily expanded, the system successfully quantified EGFR plasmid solutions, exhibiting a dynamic linear range of 105 and a detection limit of 10 copies per reaction. To further validate its performance, we employed the dPCR platform for quantitative detection of BCR-ABL1 mutation gene fragments, where its performance was compared against the QuantStudio 3D, and the self-heating dPCR system demonstrated similar analytical accuracy to the commercial dPCR system. Notably, the individual chip is produced on a semiconductor manufacturing line, benefiting from mass production capabilities, so the chips are cost-effective and conducive to widespread adoption and accessibility.

Increased lactate in AML blasts upregulates TOX expression, leading to exhaustion of CD8+ cytolytic T cells.

Recently, the role of lactate as merely an end product of cancer cell metabolism has been reassessed. Lactate has been implicated in more biological processes than previously understood and drives tumor progression. Here, we demonstrated that the bone marrow lactate concentrations in acute myeloid leukemia (AML) patients were substantially higher than those in their healthy control counterparts. Moreover, AML blasts from bone marrow expressed significantly higher lactate dehydrogenase-A (LDHA) levels. Further studies revealed that LDHA expression was regulated through the HIF1α pathway. Elevated lactate levels were indicative of alterations in CD8+ T cell cytolytic phenotype and activity. An in vitro study showed that the lactate treatment group had significantly higher percentages of CD8+ TEM and CD8+ TEMRA cells as well as higher PD-1 expression in these cells than the control group. Lactate induced the loss of the effector function of CD8+ T cells by altering lytic granule exocytosis. T cell dysfunction is characterized by an increase in terminally differentiated phenotypes, sustained expression of PD-1, and accelerated decline of cytolytic competence. Moreover, the TOX gene was found to be correlated with lactate production and implicated in CD8+ T cell dysfunction. AML patients in complete remission after chemotherapy had markedly lower lactate concentrations, reduced CD8+ TEM and CD8+ TEMRA cells and PD-1 expression, and increased perforin and granzyme B. However, no difference was found in the relapsed patients. The study presented here has established lactate as a predictive biomarker for patient response to antitumor therapies and demonstrated that targeting this gene in AML patients could be a meaningful precision therapeutic strategy.

Soluble CD40 ligand inhibits the growth of non-Hodgkin's lymphoma cells through the JNK signaling pathway.

The incidence of non-Hodgkin's lymphoma (NHL) has been increasing annually and has become a serious threat to human health. However, the pathogenesis of NHL remains unclear. The present study aimed to investigate the effect of soluble CD40 ligand (sCD40L) on NHL cells and its underlying mechanism. Cell Counting kit-8 assay and flow cytometry apoptosis experiments were conducted to investigate the effects of sCD40L on cell proliferation and apoptosis. Western blotting was performed to detect the protein expression levels of BAX, Bcl-2, ERK, p-ERK, JNK, p-JNK, p38, p-p38 and c-JUN. The results of the present study demonstrated that exogenous sCD40L significantly inhibited the proliferation and promoted the apoptosis of Raji and CA46 cells. Additionally, exogenous sCD40L promoted the apoptosis of lymphoma cells by activating the JNK signaling pathway.

Clonal expansion of bone marrow CD8+ T cells in acute myeloid leukemia patients at new diagnosis and after chemotherapy.

CD8+ T cells are crucial adaptive immune effectors and express receptors (T cell receptors, TCRs) that specifically recognize and eradicate tumor cells. The diversity of the TCR repertoire is generated by specialized genetic diversification mechanisms, which lead to an extremely variable TCR repertoire that is capable of recognizing a wide range of antigens. However, the variations in CD8+ TCR diversity and their clinical implications in acute myeloid leukemia (AML) patients remain unknown. CD8+ T cells were enriched from 10 healthy donors and 31 AML patients at diagnosis and after chemotherapy, and TCRß deep sequencing was performed to analyze CD8+ T cell clonal expansion and TCR repertoire diversity. Diminished TCR repertoire diversity and increased T cell clone expansion were noted in the bone marrow of AML patients. In relapsed patients, T cells were found to be more clonally expanded after chemotherapy than at new diagnosis. Moreover, significantly more expanded TCRß clonotypes were noted in CD8+ PD-1+ T cells than in CD8+ PD-1- T cells regardless of the time of examination. Our systematic T cell repertoire analysis may help better characterize CD8+ T cells before and after chemotherapy in AML, which may provide insights into therapeutic strategies for hematological malignancies.

[Effects of Soluble CD40 Ligand on Non-Hodgkin Lymphoma Cells and Its Relative Mechanism].

OBJECTIVE: To explore the effect of soluble CD40 ligand (sCD40L) on the proliferation, apoptosis, and cell cycle of human non-Hodgkin lymphoma (NHL) cells, and analyze its possible mechanism. METHODS: NHL CA46 cell and Raji cell were treated with different concentrations of sCD40L for 48 h, CCK-8 was used to detect the effect of sCD40L on cell proliferation in vitro, flow cytometry on apoptosis and cycle of NHL cells, and Western blot on the expression of PTEN, BCL-2, and BAX in NHL cells. RESULTS: Compared with the control group, 4 and 8 µg/ml sCD40L could significantly inhibit the proliferation of lymphoma Raji cell and CA46 cell (P<0.05). The test results of flow cytometry showed that 4 µg/ml sCD40L could significantly promote the apoptosis of CA46 and Raji cells, and significantly inhibit the S phase proportions (P<0.05). Western blot results showed that sCD40L could promote the expression of PTEN and BAX, while inhibit the expression of BCL-2 (P<0.05). CONCLUSION: sCD40L can promote the apoptosis and inhibit the proliferation of NHL cells through the PTEN signaling pathway.

[Construction and Identification of Acute Myeloid Leukemia NOD/SCID Mouse Model by Tail Vein Injection of THP-1 Cells].

OBJECTIVE: To construct NOD/SCID mouse leukemia model by using THP-1 cells. METHODS: Eighteen female NOD/SCID mice aged 3 to 4 weeks were randomly divided into control group, model group A and model group B (6 in each group). Before inoculation, each mouse was intraperitoneally injected with cyclophosphamide 2 mg/(kg·d) for 2 d, and the mice in model groups were inoculated with cells within 24 h after pretreatment. The mice in model group were inoculated with THP-1 cell suspension in logarithmic growth phase by 1×107 cells/group (group A) and 5×106 cells/group (group B), the mice in the control group were injected with the same amount of normal saline in the tail vein. The general situation was observed, blood routine test and peripheral blood leukocyte classification were performed at 7, 14, 21, 28 d of inoculation before the pre-treatment, and at the time sacrifice. Before dying, tissue of mice were collected and histological examination was performed. RESULTS: Pilereation, droopiness and hypkinesia could be observed from d 7 and d 10 of inoculation cells in model group. Compared with the control group, the body weight of the mice in model group A and B decreased significantly after 21 days of inoculation (P<0.01), and the white blood cell counts increased significantly after 28 days of modeling (P<0.01). Among them, the above-mentioned presentation in inoculation of 1×107 group A was the most significant. Histopathological sections showed diffuse infiltration of leukemia cells in the spleen of the model group. The immunohistochemistry results indicated that the leukemia cells were positive for anti-human CD13, which confirmed the successful establishment of the model. CONCLUSION: After pretreatment with intraperitoneal injection of CTX in NOD/SCID mice, the injection of 1×107 or 5×106 THP-1 cells in tail vein of each mouse can successfully construct an acute myeloid leukemia animal model. The tumor formation is more much faster by injection of high concentration THP-1 cells.

Raised CD40L expression attenuates drug resistance in Adriamycin-resistant THP-1 cells.

Acute myeloid leukemia is a common hematological malignancy that often exhibits strong drug resistance when treated using conventional chemotherapy. Although numerous studies have been carried out to develop methods of overcoming drug resistance, the results have generally been unsatisfactory. CD40 ligand (CD40L) has been shown to improve the sensitivity of cancer cells to drug treatment. In the present study, Adriamycin (ADM)-resistant human monocytic THP-1 cells (THP-1/A cells) were developed by incubating THP-1 cells with increasing concentrations of ADM. Cells were transfected with CD40L vectors to explore the potential involvement of CD40L in regulating multidrug resistance (MDR) in cancer. Cell proliferation and viability were measured using the Cell Counting Kit-8 assay; cell apoptosis was evaluated by flow cytometry, trypan blue staining and caspase-3 activity; and the expression of MDR-associated protein 1 (MRP1) and permeability glycoprotein (P-gp) was analyzed using western blotting. The results revealed that the protein expression levels of MRP1 and P-gp were downregulated by raised CD40L expression and that the combination of raised CD40L expression with daunorubicin (DNR), a drug from which ADM is derived, significantly increased the extent of cell apoptosis, indicating that drug resistance was effectively attenuated by CD40L. Collectively, these results suggested that CD40L may contribute towards reducing DNR resistance in THP-1/A cells.

Cetuximab enhances the efficiency of irinotecan through simultaneously inhibiting the MAPK signaling and ABCG2 in colorectal cancer cells.

BACKGROUND: The present study sought to investigate the combined effects of cetuximab and irinotecan on colorectal cancer cells as well as the mechanisms underlying their anti-cancer effects. MATERIAL AND METHODS: High performance liquid chromatography, Hoechst staining assay, and western blotting analysis were used to detect intracellular drug concentrations, cell apoptosis, and protein expression in the presence of cetuximab, irinotecan, and the combination of both. RESULTS: Cetuximab was found to increase intracellular concentrations of irinotecan as well as cytotoxicity by inhibiting the epidermal growth factor receptor and, by extension, the downstream RAS-RAF-MEK-ERK signaling pathway. Cetuximab therefore induced apoptosis and improved the effect of irinotecan in colorectal cancer cells. It was also shown that cetuximab inhibited the drug efflux activity of ABCG2. In combination with irinotecan, cetuximab can both significantly induce cell apoptosis by inhibiting the RAS-RAF-MEK-ERK signaling pathway and improve the effects of irinotecan by decreasing drug efflux through the inhibition of ABCG2. CONCLUSION: These features contribute to its anti-cancer potential.

CD40L inhibits cell growth of THP-1 cells by suppressing the PI3K/Akt pathway.

INTRODUCTION: Acute myeloid leukemia (AML), the hematological malignant tumor with high mortality, is still difficult to treat. CD40L is a type II transmembrane protein, which has been reported to have the potential to inhibit growth of some cancer cells. MATERIALS AND METHODS: In order to determine the role of CD40L on AML-M5 cell line THP-1, we overexpressed CD40L in the cells using a lentiviral vector system (pHBLV-CMVIE-Zs Green-T2A-puro vector); overexpression was confirmed by the detection of green fluorescent protein and CD40L protein expression. RESULTS: Cellular apoptosis, proliferation, and cycle assays showed that CD40L could promote the apoptosis of, suppress the proliferation of, and stimulate the arrest of the G1/S phase of THP-1 cells. Finally, the protein expression of P53, Bax/Bcl-2, cyclinD1, PCNA, PTEN, and p-Akt illustrated that CD40L may partly influence cell growth of THP-1 cells through those genes, which was confirmed by immunohistochemistry and a PI3K/Akt activator. CONCLUSION: Taken together, CD40L could inhibit cell growth of THP-1 cells through the PI3K/Akt pathway, indicating that the overexpression of CD40L may be a potential target to treat the AML-M5 disease.

Regulatory effect of Act1 on the BAFF pathway in B-cell malignancy.

The aim of the present study was to ascertain whether nuclear factor (NF)-κB Activator 1 (Act1) was involved in B cell-activating factor (BAFF) regulation in B-cell malignancy. The human B-cell malignancy cell lines Raji, Daudi and BALL-1 were cultured and the expression of BAFF receptor (BAFF-R) mRNA and protein was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. NF-κB signaling was also assessed using western blotting. Act1 silencing was performed using Act1 small interfering RNA. BAFF-R levels were assessed using flow cytometry. It was demonstrated that BAFF-R was upregulated in all three cell lines and RT-qPCR, and western blotting confirmed these results. Act1 overexpression was demonstrated to induce BAFF-R upregulation, whereas Act1 knockdown resulted in BAFF-R downregulation. Furthermore, the NF-κB pathway was activated by Act1 overexpression and inhibited following Act1 knockdown. The results of the present study demonstrated that Act1 can regulate BAFF via targeting NF-κB signaling, which suggests that Act1 may be a promising therapeutic target for the treatment of B-cell malignancy.

Functional Variant rs4442975 Modulating FOXA1 Binding Affinity Can Influence Bone Marrow Suppression during Neoadjuvant Chemotherapy for Luminal A Type Breast Cancer.

The expression of the transcription factor FOXA1 is associated with the prognosis of estrogen receptor (ER)-positive breast cancer, and the genetic variant rs4442975 can affect FOXA1 function. Therefore, we investigated the association between rs4442975 and the efficacy of neoadjuvant chemotherapy for luminal A type breast cancer and evaluated its toxic side effects in a Chinese population. One hundred seventy-five patients with luminal A type breast cancer receiving neoadjuvant chemotherapy with a combination protocol of epirubicin and docetaxel (ET protocol) were enrolled in the study. Genotyping was performed in a randomized manner to identify candidate genetic variants. Unconditional logistic regression analysis was used to analyze the association of the variant with the efficacy and side effects of neoadjuvant chemotherapy. The results did not reveal any positive association with the efficacy of neoadjuvant chemotherapy, with an odds ratio (OR) of 0.73 (95% confidence interval = 0.27-1.94) in the additive model. However, analysis of the toxic side effects of neoadjuvant chemotherapy showed that rs4442975 was associated with bone marrow suppression, with an OR of 0.38 (95% confidence interval = 0.17-0.73, p = 0.005) in the dominant model. In summary, the functional genetic variant rs4442975 was associated with bone marrow suppression during neoadjuvant chemotherapy for luminal A type breast cancer. These results may help establish reliable molecular markers for predicting the prognosis of personalized treatment for luminal A type breast cancer and thereby contribute to the development of appropriate therapies.

H19 contributes to poor clinical features in NSCLC patients and leads to enhanced invasion in A549 cells through regulating miRNA-203-mediated epithelial-mesenchymal transition.

Recent studies have demonstrated that the overexpression of H19 may contribute towards development of tumorigenesis in various types of cancer. To investigate the role of H19 in the development of non-small cell lung cancer (NSCLC), 76 NSCLC tissues samples and their adjacent normal tissue samples were collected. Expression level of H19, and its association with clinicopathological features and overall survival was analyzed. It was found that compared with normal adjacent tissues, H19 expression was elevated in NSCLC tissues along with a decreased miR-203 expression level. It was also found that patients who were in advanced clinical stages had a higher H19 and a lower miR-203 expression compared to normal tissues. The overall survival time of patients with higher H19 expression was shorter compared with the lower H19 expression group. Upregulation of A549 enhanced cell proliferation and promoted invasion. Overexpression of H19 stimulated the epithelial-mesenchymal transition (EMT) process in lung cancer cells and demonstrated typical morphological characteristics of EMT. The level of mesenchymal marker protein, such as Vimentin and SNAI1 increased; while CDH1 protein level decreased. Also, H19 negatively regulated miR-203. Inhibition of H19 attenuated miR-203 induced EMT process. Upregulation of H19 contributes to poor clinical features in patients with NSCLC, induces occurrence of EMT, promotes proliferation and stimulates cell invasion in NSCLC cell line through regulating miRNA-203 mediated EMT.