Preclinical study and phase II trial of adapting low-dose radiotherapy to immunotherapy in small cell lung cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) provide modest but unsatisfactory benefits for extensive-stage small cell lung cancer (ES-SCLC). Developing strategies for treating ES-SCLC is critical. METHODS: We preliminarily explored the outcomes of salvage low-dose radiotherapy (LDRT) plus ICI on refractory SCLC patients. Next, we evaluated the combinational efficacy in murine SCLC. The tumor immune microenvironment (TIME) was analyzed for mechanistic study. Subsequently, we conducted a multicenter, prospective phase II trial that administered concurrent thoracic LDRT plus chemoimmunotherapy to treatment-naive ES-SCLC patients (MATCH trial, NCT04622228). The primary endpoint was confirmed objective response rate (ORR), and the key secondary endpoints included progression-free survival (PFS) and safety. FINDINGS: Fifteen refractory SCLC patients treated with LDRT plus ICI were retrospectively reviewed. The ORR was 73.3% (95% confidence interval [CI], 44.9-92.2). We identified a specific dose of LDRT (15 Gy/5 fractions) that exhibited growth retardation and improved survival in murine SCLC when combined with ICIs. This combination recruited a special T cell population, TCF1+ PD-1+ CD8+ stem-like T cells, from tumor-draining lymph nodes into the TIME. The MATCH trial showed a confirmed ORR of 87.5% (95% CI, 75.9-94.8). The median PFS was 6.9 months (95% CI, 5.4-9.3). CONCLUSIONS: These findings verified that LDRT plus chemoimmunotherapy was safe, feasible, and effective for ES-SCLC, warranting further investigation. FUNDING: This research was funded by West China Hospital (no. ZYJC21003), the National Natural Science Foundation of China (no. 82073336), and the MATCH trial was fully funded by Roche (China) Holding Ltd. (RCHL) and Shanghai Roche Pharmaceuticals Ltd. (SRPL).

CDK4/6 inhibitor abemaciclib combined with low-dose radiotherapy enhances the anti-tumor immune response to PD-1 blockade by inflaming the tumor microenvironment in Rb-deficient small cell lung cancer.

Background: Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have shown significant activity against several solid tumors by reducing the phosphorylation of the canonical CDK4/6 substrate retinoblastoma (Rb) protein, while the anti-tumor effect of CDK4/6 inhibitors on Rb-deficient tumors is not clear. Most small cell lung cancers (SCLCs) are Rb-deficient and show very modest response to immune checkpoint blockade (ICB) despite recent advances in the use of immunotherapy. Here, we aimed to investigate the direct effect of CDK4/6 inhibition on SCLC cells and determine its efficacy in combination therapy for SCLC. Methods: The immediate impact of CDK4/6 inhibitor abemaciclib on cell cycle, cell viability and apoptosis in four SCLC cell lines was initially checked. To explore the effect of abemaciclib on double-strand DNA (ds-DNA) damage induction and the combination impact of abemaciclib coupled with radiotherapy (RT), western blot, immunofluorescence (IF) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. An Rb-deficient immunocompetent murine SCLC model was established to evaluate efficacy of abemaciclib in combination therapy. Histological staining, flow cytometry analysis and RNA sequencing were performed to analyze alteration of infiltrating immune cells in tumor microenvironment (TME). Results: Here, we demonstrated that abemaciclib induced increased ds-DNA damage in Rb-deficient SCLC cells. Combination of abemaciclib and RT induced more cytosolic ds-DNA, and activated the STING pathway synergistically. We further showed that combining low doses of abemaciclib with low-dose RT (LDRT) plus anti-programmed cell death protein-1 (anti-PD-1) antibody substantially potentiated CD8+ T cell infiltration and significantly inhibited tumor growth and prolonged survival in an Rb-deficient immunocompetent murine SCLC model. Conclusions: Our results define previously uncertain DNA damage-inducing properties of CDK4/6 inhibitor abemaciclib in Rb-deficient SCLCs, and demonstrate that low doses of abemaciclib combined with LDRT inflame the TME and enhance the efficacy of anti-PD-1 immunotherapy in SCLC model, which represents a potential novel therapeutic strategy for SCLC.

Stem-like T cells and niches: Implications in human health and disease.

Recently, accumulating evidence has elucidated the important role of T cells with stem-like characteristics in long-term maintenance of T cell responses and better patient outcomes after immunotherapy. The fate of TSL cells has been correlated with many physiological and pathological human processes. In this review, we described present advances demonstrating that stem-like T (TSL) cells are central players in human health and disease. We interpreted the evolutionary characteristics, mechanism and functions of TSL cells. Moreover, we discuss the import role of distinct niches and how they affect the stemness of TSL cells. Furthermore, we also outlined currently available strategies to generate TSL cells and associated affecting factors. Moreover, we summarized implication of TSL cells in therapies in two areas: stemness enhancement for vaccines, ICB, and adoptive T cell therapies, and stemness disruption for autoimmune disorders.

Personalized neoantigen-pulsed dendritic cell vaccines show superior immunogenicity to neoantigen-adjuvant vaccines in mouse tumor models.

Development of personalized cancer vaccines based on neoantigens has become a new direction in cancer immunotherapy. Two forms of cancer vaccines have been widely studied: tumor-associated antigen (including proteins, peptides, or tumor lysates)-pulsed dendritic cell (DC) vaccines and protein- or peptide-adjuvant vaccines. However, different immune modalities may produce different therapeutic effects and immune responses when the same antigen is used. Therefore, it is necessary to choose a more effective neoantigen vaccination method. In this study, we compared the differences in immune and anti-tumor effects between neoantigen-pulsed DC vaccines and neoantigen-adjuvant vaccines using murine lung carcinoma (LL2) candidate neoantigens. The enzyme-linked immunospot (ELISPOT) assay showed that 4/6 of the neoantigen-adjuvant vaccines and 6/6 of the neoantigen-pulsed DC vaccines induced strong T-cell immune responses. Also, 2/6 of the neoantigen-adjuvant vaccines and 5/6 of the neoantigen-pulsed DC vaccines exhibited potent anti-tumor effects. The results indicated that the neoantigen-pulsed DC vaccines were superior to the neoantigen-adjuvant vaccines in both activating immune responses and inhibiting tumor growth. Our fundings provide an experimental basis for the selection of immune modalities for the use of neoantigens in individualized tumor immunotherapies.

ATP-responsive mitochondrial probes for monitoring metabolic processes of glioma stem cells in a 3D model.

The metastatic cascade of cancer stem cells (CSCs) is always accompanied by elevated levels of adenosine triphosphate (ATP) as well as the alterntion of energy metabolism to support their differentiation and migration. Here we propose a 3D microfluidic tumor model coupled with an ATP-responsive mitochondrial probe (AMP) for investigation of metabolic processes of glioma stem cells (GSCs). The 3D tumor model has a middle matrix gel microchannel mimicking the extracellular matrix (ECM), which is sandwiched between a GSC culture chamber and a stimulation chamber. The AMPs consist of structure-switching ATP aptamers and triphenylphosphonium (TPP)-conjugated peptide nucleic acids (PNAs). Under TGF-ß stimulation, invasive migration of GSCs accompanied by a high ATP level and spindle mesenchymal morphologies is observed due to the epithelial-to-mesenchymal transition (EMT). Moreover, acidic stress can keep GSCs in a low-energy state, while long-term low pH stimulation screens out more malignant glioma cells. This AMP-assisted 3D microfluidic tumor model provides a tremendous opportunity for studying the biological properties of CSCs.

Reconstituting Glioma Perivascular Niches on a Chip for Insights into Chemoresistance of Glioma.

In this work, we report the direct diagnosing chemoresistance of glioma stem cells (GSCs) during chemotherapy on a biomimetric microsystem that reconstitutes glioma perivascular niches on a chip. Glioma stem cells and endothelial cells were specially cocultured onto the biomimetric system to precisely control stem cell coculture for the proof-of-principle studies. The expression levels of 6- O-methylguanine was confirmed by mass spectrometer, and Bmi-1 gene was also investigated to uncover the chemoresistance of GSCs. The results demonstrated that the formation of perivascular niches effectively maintains the glioma stem cells at a pluripotent status owing to their successful cellular interactions. A stronger chemoresistance of glioma stem cells was confirmed by the formation of the GSCs neurosphere, the expression levels of 6- O-methylguanine and Bmi-1 gene. The vital role of endothelial cells in chemoresistance was demonstrated. The chemoresistance reported in this work will contribute to glioma therapy.

A dual-functional microfluidic chip for on-line detection of interleukin-8 based on rolling circle amplification.

Interleukin 8 (IL-8), also known as C-X-C motif ligand 8(CXCL8), is a proinflammatory chemokine functioned in neutrophil chemotaxis and activation. And it plays an important role in the process of glioma stem-like cell vascularization in the latest research. Herein, a dual-function microfluidic biosensor based on rolling circle amplification (RCA) was fabricated for cell culture and online IL-8 detection. A microfluidic chip was designed with two high passages connected by the vertical channels. One of the channels with immobilized capture antibody was prepared for IL-8 detection and another channel for cell culture. Immunoassays were achieved by a sandwich structure consisting of antibodies, IL-8, and aptamers. Signal amplification was mainly due to RCA and biotin-streptavidin linkage. The linear range for IL-8 was 7.5 -120pgmL-1 in this assay. Moreover, the developed method was successfully applied to detect the IL-8 in tumor-derived endothelial cells (TDEC) and Human Umbilical Vein Endothelial cells (HUVEC) under chemical hypoxia condition. Semi-quantitative detection of IL-8 consumption in HUVEC cells in low oxygen condition was also achieved. These results were in statistical agreement with those obtained by commercial assay of enzyme-linked immunoassay kit (ELISA). The microfluidic chip based biosensor reported hereby has a large prospect in the basic research and clinical diagnosis of cancer stem cell.

The pathological structure of the perivascular niche in different microvascular patterns of glioblastoma.

The perivascular niche is critical for intercellular communication between resident cell types in glioblastoma (GBM), and it plays a vital role in maintaining the glioma stem cell (GSC) microenvironment. It is shown in abundant research that different microvascular patterns exist in GBM; and it can be implied that different microvascular patterns are associated with different pathological structures in the perivascular niche. However, the pathological structure of the perivascular niche is still not clear. Here, we investigated the distribution and biological characteristics of different microvascular pattern niches (MVPNs) in GBM by detecting the expression of CD34, CD133, Nestin, α-SMA, GFAP and CD14 in the perivascular niche using multiple -fluorescence. The four basic microvascular patterns are microvascular sprouting (MS), vascular cluster (VC), vascular garland (VG), and glomeruloid vascular proliferation (GVP). By analyzing the proportion of the area of each marker in four types of formations, the results indicated that the expression of CD34, CD133 and Nestin in MS and VC was significantly lower than that in VG and GVP (P<0.05). Furthermore, the results showed that α-SMA expression different in the MS, VC, VG and GVP (P<0.05). However, the expression of GFAP and CD14 in each type of formation exhibited no significant difference (P>0.05). According to the area distributions of different markers, we mapped four precise simulation diagrams to provide an effective foundation for the accurate simulation of glioblastoma in vitro.

Quantitative determination of VEGF165 in cell culture medium by aptamer sandwich based chemiluminescence assay.

In this work, we have developed a sensitive and selective chemiluminescence (CL) assay for vascular endothelial growth factor (VEGF165) quantitative detection based on two specific VEGF165 binding aptamers (Apt). VEGF is a predominant biomarker in cancer angiogenesis, and sensitive detection method of VEGF are highly demanded for both academic study and clinical diagnosis of multiple cancers. In our experiment, VEGF165 was captured in a sandwich structure assembled by two binding aptamers, one capture aptamer was immobilized on streptavidin-coated magnetic beads (MBs) and another VEGF-binding aptamer was labeled by biotin for further phosphatase conjunction. After Apt-VEGF-Apt sandwich was formed on MBs surface, alkaline phosphatase (ALP) was modified to the second aptamer to catalyze CL reaction. By applying 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2-adamantane) (AMPPD) as CL substrate, strong signal intensity was achieved. VEGF165 content as low as 1ng/mL was detected in standard spiked samples by our assay, and linear range of working curve was confirmed from 1 to 20ng/mL. Then our method was successfully applied for cell culture medium analysis and on-chip hypoxic HepG2-HUVEC co-culture model study with excellent accuracy equal to ELISA Kit. Our developed assay demonstrated an outstanding performance in VEGF165 quantification and may be further extended to clinical testing of important biomarkers as well as probing microchip cell culture model.

Dynamic imaging of MYC and CDKN1A mRNAs as an indicator of cell G1-phase arrest.

The identification of G1-phase arrest requires a dynamic capturing method. In this work, we reported an indicator based on MYC and CDKN1A mRNA imaging to visualize G1-phase cycle arrest. A typical G1-phase arrest imaging is shown as a green-to-red conversion by fluorescence molecular beacons indicating the change as down-regulating of MYC and up-regulating of CDKN1A mRNA. We performed G1-phase arrest imaging on proliferated cancer cells inhibited by TMPyP4 and explore the role of HIF-1α in the proliferation inhibition effect.

A DNA-directed covalent conjugation fluorescence probe for in vitro detection of functional matrix metalloproteinases.

Matrix metalloproteinases (MMPs) have been considered to contribute to the progression of tumorigenesis and tumor invasion; MMP-9 in particular, has been regarded as a priority target in cancer treatment due to its massive up-regulation in malignant tissues and its ability to degrade type IV collagen. In this work, we employed a DNA-directed covalent conjugation method to design a fluorescence probe for in vitro detection of functional matrix metalloproteinases, by which a nitrilotriacetic acid (NTA)-modified DNA probe can combine with the Zn2+ in the active site of MMPs, and then a molecule beacon (MB) modified FITC and BHQ1 can open to bond with their complementary base, NTA-modified DNA. We can evaluate the amount of MMPs in the medium according to the fluorescence intensity. The detection procedure can be finished in 30 min with good selectivity, cheap reagents and easy preparation. All the results and the amount of secreted MMPs under three different cell culture conditions are in accordance with previous reports. Satisfactory results are obtained. Furthermore, owing to the importance of MMP-9, we designed an approach to achieve the desired selectivity and specificity of our work, using dual amplification for improving fluorescence intensity based on RCA to detect the amount of MMP-9.

A non-invasive genomic diagnostic method for bladder cancer using size-based filtration and microchip electrophoresis.

Bladder cancer (BC) cells spontaneously exfoliated in the urine of patients with BC. Detection of exfoliated tumor cells has clinical significance in cancer therapy because it would enable earlier non-invasive screening, diagnosis, or prognosis of BC. In this research, a method for analyzing genetic abnormalities of BC cells collected from urine samples was developed. Target BC cells were isolated by filtration. To find conditions that achieve high cell recovery, we investigated the effects of filter type, concentration of fixative, and flow rate. Cells captured on the filter membrane were completely retrieved within 15s. Selected genes for genomic analysis, mutated genes (FGFR3, TERT and HRAS) and methylated genes (ALX4, RALL3, MT1A, and RUNX3) were amplified by polymerase chain reaction (PCR), and subsequently, were identified by microchip electrophoresis (MCE). Analysis by MCE reduces the risk of contamination, sample consumption, and analysis time. Our developed approach is economical, effectively isolates cancer cells, and permits flexible molecular characterization, all of which make this approach a promising method for non-invasive BC detection.

A comparative study of three different nucleic acid amplification techniques combined with microchip electrophoresis for HPV16 E6/E7 mRNA detection.

Research towards nucleic acid amplification technologies for detection of human papillomavirus (HPV) 16 E6/E7 mRNA was carried out in combination with microchip electrophoresis (MCE). The approaches of nucleic acid sequence based amplification (NASBA), one-step RT-PCR and two-step RT-PCR were successfully developed. NASBA was a simple enzymatic reaction, which directly amplified HPV16 mRNA by isothermal amplification, leaving out the complex and tedious operation. One-step RT-PCR simplified the amplification step, while two-step RT-PCR was more sensitive and less vulnerable to the interference. Furthermore, instead of gel electrophoresis, microchip electrophoresis (MCE) for RNA assay was employed to realize high-throughput and rapid analysis. Finally, the results show that PCR-based or NASBA-based mRNA tests are valuable for HPV mRNA assay, which can be potentially applied for clinical diagnosis and prognosis of cervical and other anogenital carcinoma.

Assay of multiplex proteins from cell metabolism based on tunable aptamer and microchip electrophoresis.

A simple and rapid method for multiplex protein assay based on tunable aptamer by microchip electrophoresis has been developed. Different lengths of aptamers can modulate the electrophoretic mobility of proteins, allowing the protein molecules to be effectively separated in hydroxyethyl cellulose buffer with 1.00 mM magnesium ion. A non-specific DNA was exploited as an internal standard to achieve the quantitative assay and to reduce the interference. A fluorescence dye SYBR gold was exploited to improve the sensitivity and to suppress the interference from sample matrix. Under optimum conditions, quantitative assay of PDGF-BB (R(2)=0.9986), VEGF165 (R(2)=0.9909), and thrombin (R(2)=0.9947) were achieved with a dynamic range in the 5.00-150.0 nM and RSDs in the 5.87-16.3% range. The recoveries were varied from 83.6% to 113.1%. Finally, the proposed method was successfully applied to analyze cell secretions, and then the concentration of PDGF-BB and VEGF165 were detected from 5.15 nM to 2.03 nM, and 3.14 to 2.53 nM, respectively, indicating the established method can be used to analyze cell secretions.

High-throughput and automatic typing via human papillomavirus identification map for cervical cancer screening and prognosis.

A novel human papillomavirus (HPV) typing assay for cervical cancer screening and prognosis was developed by the combination of restriction fragment length polymorphism (RFLP) and microchip electrophoresis (MCE) to achieve higher levels of sensitivity and throughput. The detection limit of 2 × 10(2) copies, high sensitivity and typing accuracy on the account of PCR-RFLP-MCE method guarantee the successful diagnosis results of 4-fold higher infection rate over cytologic tests. From clinical samples, eleven kinds of HPV types were identified with a good compatibility degree of over 90%. The described method showed good reliability in clinical samples and provided a promising alternative for pathological studies at the molecular level.