Synergy of Front-Surface Energy-Level Gradient and Lattice Anchoring Effect for Enhancing Perovskite Solar Cell Performance.

A front surface gradient of the absorber valence band can effectively reduce the open-circuit voltage (VOC ) loss of perovskite solar cells by suppressing the minority carrier concentration near the front surface. However, the existing method is limited to the one-step fabrication process, resulting in underachieved photon harvesting and power conversion efficiency (PCE). To solve the problem, ZnCd-based alloy quantum dots (QDs) are utilized to create a valence-band-maximum gradient at the front surface of a two-step processed FAPbI3 absorber. This design significantly enhances VOC without requiring surface passivation. Furthermore, it is demonstrated that reducing the QD-perovskite lattice mismatch while maintaining QD's energy levels mitigates nonradiative recombination without compromising the front surface gradient effect. As a result, normal-structured perovskite solar cells achieve a VOC equivalent to 93% of the Schockley-Queisser limit and a PCE of 24.37%.

IL6-mediated inflammatory loop reprograms normal to epithelial-mesenchymal transition+ metastatic cancer stem cells in preneoplastic liver of transforming growth factor beta-deficient ß2-spectrin+/- mice.

Hepatocellular carcinoma (HCC) is the second-leading cause of cancer-related deaths worldwide with a poor survival rate. As many as 40% of HCCs are clonal, with alteration of key tumor-suppressor pathways in stem cells as the primary cause of HCC initiation. However, mechanisms that generate metastatic stem cells in preneoplastic liver tissue are not well understood. We hypothesized that chronic inflammation is a major driver of the transformation of genetically defective liver stem cells (LSCs) into highly metastatic liver cancer cells in premalignant liver tissue. We developed models of chronic inflammation in wild-type (WT) and ß2-spectrin (ß2SP)+/- (SPTBN1) mice. CD133+ LSCs derived from preneoplastic livers of ß2SP+/- mice treated with interleukin-6 (pIL6; IL6 ß2SP+/- LSCs) were highly tumorigenic and metastatic, whereas those derived from WT mice treated with pIL6 (IL6 WT LSCs) had significantly less proliferation and no tumorigenic properties. IL6 ß2SP+/- LSCs not only exhibited nuclear localization of Twist and Slug, markers of epithelial-mesenchymal transition (EMT), but also constitutive activation of nuclear factor kappa B (NFκB; RELA). Knockdown of NFκB decreased the EMT phenotypes and metastatic capacity of these cells. NFκB in IL6 ß2SP+/- LSCs was activated by transforming growth factor ß (TGFß)-activated kinase 1 (TAK1; MAP3K7), which is associated with poor survival in HCC and interleukin-6 (IL6) expression. The amount of constitutively activated NFκB increased dramatically from normal to cirrhotic to HCC tissues from human patients. CONCLUSION: IL6-mediated inflammation programs constitutive activation of the TAK1-NFκB signaling cascade in CD133+ LSCs, and this program interacts with deficient TGFß signaling, thereby accelerating the transformation of normal LSCs to metastatic cancer stem cells (mCSCs). Indeed, this study delineates the development of EMT-positive mCSCs in HCC-free liver tissue upon chronic inflammation. (Hepatology 2017;65:1222-1236).

Mutant p53 in concert with an interleukin-27 receptor alpha deficiency causes spontaneous liver inflammation, fibrosis, and steatosis in mice.

UNLABELLED: The cellular and molecular etiology of unresolved chronic liver inflammation remains obscure. Whereas mutant p53 has gain-of-function properties in tumors, the role of this protein in liver inflammation is unknown. Herein, mutant p53(R172H) is mechanistically linked to spontaneous and sustained liver inflammation and steatosis when combined with the absence of interleukin-27 (IL27) signaling (IL27RA), resembling the phenotype observed in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) patients. Indeed, these mice develop, with age, hepatocyte necrosis, immune cell infiltration, fibrosis, and micro- and macrosteatosis; however, these phenotypes are absent in mutant p53(R172H) or IL27RA(-/-) mice. Mechanistically, endothelin A receptor (ETAR)-positive macrophages are highly accumulated in the inflamed liver, and chemical inhibition of ETAR signaling reverses the observed phenotype and negatively regulates mutant p53 levels in macrophages. CONCLUSION: The combination of mutant p53 and IL27RA(-/-) causes spontaneous liver inflammation, steatosis, and fibrosis in vivo, whereas either gene alone in vivo has no effects on the liver.

Interleukin-30 (IL27p28) alleviates experimental sepsis by modulating cytokine profile in NKT cells.

BACKGROUND & AIMS: Sepsis is an acute systemic inflammatory response to infection associated with high patient mortality (28-40%). We hypothesized that interleukin (IL)-30, a novel cytokine protecting mice against liver injury resulting from inflammation, would generate a protective effect against systemic inflammation and sepsis-induced death. METHODS: Sepsis was induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). The inhibitory effects of IL-30 on septic inflammation and associated therapeutic effects were determined in wild-type, IL30 (p28)(-/-), IL10(-/-), and CD1d(-/-) mice. RESULTS: Mice treated with pIL30 gene therapy or recombinant IL-30 protein (rIL30) were protected from LPS-induced septic shock or CLP-induced polymicrobial sepsis and showed markedly less liver damage and lymphocyte apoptosis than control septic mice. The resulting reduction in mortality was mediated through attenuation of the systemic pro-inflammatory response and augmentation of bacterial clearance. Mice lacking IL-30 were more sensitive to LPS-induced sepsis. Natural killer-like T cells (NKT) produced much higher levels of IL-10 and lower levels of interferon-gamma and tumor necrosis factor-alpha in IL-30-treated septic mice than in control septic mice. Likewise, deficiency in IL-10 or NKT cells abolished the protective role of IL-30 against sepsis. Furthermore, IL-30 induced IL-10 production in purified and LPS-stimulated NKT cells. Blocking IL-6R or gp130 inhibited IL-30 mediated IL-10 production. CONCLUSIONS: IL-30 is important in modulating production of NKT cytokines and subsequent NKT cell-mediated immune regulation of other cells. Therefore, IL-30 has a role in prevention and treatment of sepsis via modulation of cytokine production by NKT.

Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy.

Electroporation improves the anti-tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation-mediated chemotherapeutics with interleukin 12 (IL-12) plasmid DNA produces a strong yet safe anti-tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL-12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL-12 gene therapy for long-term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro-chemo-gene therapy (ECGT) with IL-12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression via an antitumour immune response in all tested histotypes except for sarcomas, and these treatments can quickly eradicate or debulk large squamous cell carcinomas. The versatility of ECGT allows for response-based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti-tumour response for successfully affecting not only the treated tumours, but also non-treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens.

Cell-surface Vimentin: A mislocalized protein for isolating csVimentin(+) CD133(-) novel stem-like hepatocellular carcinoma cells expressing EMT markers.

Recent advances in cancer stem cell biology have shown that cancer stem-like cells with epithelial-mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem-like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem-like cells, but none has been identified for isolating cancer stem-like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem-like cancer cells with EMT phenotype, by using a specific CSV-binding antibody, 84-1. Using this antibody, we purified the CSV-positive, CD133-negative (csVim(+) CD133(-) ) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim(+) CD133(-) cells have stem-like properties similar to csVim(-) CD133(+) population. Our investigation further revealed that the csVim(+) CD133(-) cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E-cadherin. The csVimentin-negative CD133-positive stem cells do not have any EMT phenotypes. csVim(+) CD133(-) cells exhibited more aggressively metastatic in livers than csVim(-) CD133(+) cells. Our findings indicate that csVim(+) CD133(-) cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.

Immune checkpoint regulator PD-L1 expression on tumor cells by contacting CD11b positive bone marrow derived stromal cells.

BACKGROUND: Expression of programmed cell death ligand 1 (PD-L1) is an important process by which tumor cells suppress antitumor immunity in the tumor microenvironment. Bone marrow (BM)-derived immune cells are an important component of the tumor microenvironment. However, the link between PD-L1 induction on tumor cells and communication with BM cells is unknown. RESULTS: This study demonstrates that BM cells have a direct effect in inducing PD-L1 expression on tumor cells, which contributes to the tumor cells' drug resistance. This novel discovery was revealed using a co-incubation system with BM cells and tumor cells. BM cells from wild-type C57BL6 mice and the immune-deficient mouse strains B-cell(-/-), CD28(-/-), perforin(-/-), and Rag2(-/-) but not CD11b(-/-) dramatically increased the expression of tumor cell surface PD-L1. This PD-L1 induction was dependent on CD11b-positive BM cells through direct contact with tumor cells. Furthermore, p38 signaling was activated in tumor cells after co-incubation with BM cells, whereas the expression of PD-L1 was remarkably decreased after co-culture of cells treated with a p38 inhibitor. The increase in PD-L1 induced by BM cell co-culture protected tumor cells from drug-induced apoptosis. CONCLUSIONS: PD-L1 expression is increased on tumor cells by direct contact with BM-derived CD11b-positive cells through the p38 signaling pathway. PD-L1 may play an important role in drug resistance, which often causes failure of the antitumor response.

CD8+T cell-specific induction of NKG2D receptor by doxorubicin plus interleukin-12 and its contribution to CD8+T cell accumulation in tumors.

BACKGROUND: Increased infiltration of CD8+T cells into tumors has a positive impact on survival. Our previous study showed that doxorubicin (Dox) plus interleukin-12 (IL-12) boosted the accumulation of CD8+T cells in tumors and had a greater antitumor effect than did either agent alone. The purpose of this study was to determine the impact of NKG2D expression on CD8+T cell infiltration and antitumor efficacy. METHODS: Tumor-bearing mice were administered Dox, IL-12 plasmid DNA, or both via intraperitoneal injection or intramuscular electroporation. The induction of NKG2D on CD8+T cells and other lymphocytes was analyzed via flow cytometry, and NKG2D-positive CD8+T cell-specific localization in tumors was determined by using immunofluorescence staining in various types of immune cell-depleted mice. RESULTS: The combination of Dox plus IL-12 specifically increased expression of NKG2D in CD8+T cells but not in other types of immune cells, including NK cells, which naturally express NKG2D. This induced NKG2D expression in CD8+T cells was associated with increased accumulation of CD8+T cells in murine tumors. Administration of NKG2D-blocking antibody or CD8+T cell-depletion antibody abrogated the NKG2D+CD8+T cell detection in tumors, whereas administration of NK cell-depletion antibody had no effect. Increased NKG2D expression in CD8+T cells was associated with increased antitumor efficacy in vivo. CONCLUSION: We conclude that Dox plus IL-12 induces NKG2D in CD8+T cells in vivo and boosts NKG2D+CD8+T-dependent antitumor immune surveillance. This discovery reveals a novel mechanism for how chemoimmunotherapy synergistically promotes T cell-mediated antitumor immune surveillance.

Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells.

BACKGROUND: Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. RESULTS: We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. CONCLUSIONS: Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

Ultrabright and stable top-emitting quantum-dot light-emitting diodes with negligible angular color shift.

Top emission can enhance luminance, color purity, and panel-manufacturing compatibility for emissive displays. Still, top-emitting quantum-dot light-emitting diodes (QLEDs) suffer from poor stability, low light outcoupling, and non-negligible viewing-angle dependence because, for QLEDs with non-red emission, the electrically optimum device structure is incompatible with single-mode optical microcavity. Here, we demonstrate that by improving the way of determining reflection penetration depths and creating refractive-index-lowering processes, the issues faced by green QLEDs can be overcome. This leads to advanced device performance, including a luminance exceeding 1.6 million nits, a current efficiency of 204.2 cd A-1, and a T95 operational lifetime of 15,600 hours at 1000 nits. Meanwhile, our design does not compromise light outcoupling as it offers an external quantum efficiency of 29.2% without implementing light extraction methods. Lastly, an angular color shift of Δu'v' = 0.0052 from 0° to 60° is achieved by narrowing the emission linewidth of quantum dots.

Cell membrane-anchored and tumor-targeted IL-12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models.

BACKGROUND: The extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted chimeric antigen receptor-T cell therapy cannot infiltrate ECM-rich tumors such as osteosarcoma. METHOD: In this study, we used RNA sequencing to assess whether the recently invented membrane-anchored and tumor-targeted IL-12-armed (attIL12) T cells, which bind cell-surface vimentin (CSV) on tumor cells, could destroy CAFs to disrupt the ECM. We established an in vitro model of the interaction between osteosarcoma CAFs and attIL12-T cells to uncover the underlying mechanism by which attIL12-T cells penetrate stroma-enriched osteosarcoma tumors. RESULTS: RNA sequencing demonstrated that attIL12-T cell treatment altered ECM-related gene expression. Immunohistochemistry staining revealed disruption or elimination of high-density CAFs and ECM in osteosarcoma xenograft tumors following attIL12-T cell treatment, and CAF/ECM density was inversely correlated with T-cell infiltration. Other IL12-armed T cells, such as wild-type IL-12-targeted or tumor-targeted IL-12-T cells, did not disrupt the ECM because this effect depended on the engagement between CSV on the tumor cell and its ligand on the attIL12-T cells. Mechanistic studies found that attIL12-T cell treatment elevated IFNγ production on interacting with CSV+ tumor cells, suppressing transforming growth factor beta secretion and in turn upregulating FAS-mediated CAF apoptosis. CAF destruction reshaped the tumor stroma to favor T-cell infiltration and tumor inhibition. CONCLUSIONS: This study unveiled a novel therapy-attIL12-T cells-for targeting CAFs/ECM. These findings are highly relevant to humans because CAFs are abundant in human osteosarcoma.

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

Interleukin-30: a novel antiinflammatory cytokine candidate for prevention and treatment of inflammatory cytokine-induced liver injury.

UNLABELLED: The liver is the major metabolic organ and is subjected to constant attacks from chronic viral infection, uptake of therapeutic drugs, life behavior (alcoholic), and environmental contaminants, all of which result in chronic inflammation, fibrosis, and, ultimately, cancer. Therefore, there is an urgent need to discover effective therapeutic agents for the prevention and treatment of liver injury, the ideal drug being a naturally occurring biological inhibitor. Here we establish the role of IL30 as a potent antiinflammatory cytokine that can inhibit inflammation-induced liver injury. In contrast, interleukin (IL)27, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the proinflammatory signal such as IL12 through interferon-gamma (IFN-γ)/signal transducer and activator of transcription 1 signaling. In animal models, administration of IL30 by way of a gene therapy approach prevents and treats both IL12-, IFN-γ-, and concanavalin A-induced liver toxicity. Likewise, immunohistochemistry analysis of human tissue samples revealed that IL30 is highly expressed in hepatocytes, yet barely expressed in inflammation-induced tissue such as fibrous/connective tissue. CONCLUSION: These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses proinflammatory cytokine-associated liver toxicity.

Luxury or normal goods? Evidence from the utilization of institutional care services for the disabled older adults in China.

Background: Nursing care is essential for older adults with disabilities. Income plays a crucial role in determining the utilization of institutional care services. Pension benefit, as the main source of income for the older adults in China's cities and towns in their later years, is an important factor influencing the utilization of institutional care services. However, there have been no consistent findings on how pension benefits affect the utilization of institutional care services for the disabled older adults. Methods: This paper utilizes data from the 2017-2018 Chinese Longitudinal Healthy Longevity Survey. We select disabled older adults aged 65 and older, living in towns and cities, and use a probit regression model to investigate the impact of pension benefits on the utilization of institutional care services by urban disabled older adults empirically. Results: The study shows that a 1% increase in pension benefits raises the probability that the urban disabled older adults use institutional care services by 0.03. It also finds that for low-income urban disabled older adults, the effect is statistically significantly positive at the 1% level; but for high-income urban disabled older adults, the effect is not statistically significant. The pension benefits significantly increase the probability for the disabled older adults who are male, financially dependent, and live in townships. In addition, the pension benefits significantly reduce the probability that children will provide care and pay for care services for their older parents. Conclusion: Institutional care service is a normal good for the urban disabled older adults, especially for low-income older adults. Therefore, higher pension benefit raises the probability of utilizing institutional care services for the urban older adults with disabilities, and this positive effect is especially pronounced for older adults who are male, financially dependent, and reside in townships. In addition, increase in the pension benefits for the disabled older adults in towns and cities reduces the burden on children by reducing the probability that children will provide care and pay for care services for the older adults.

Automated Capture and Analysis of Circulating Tumor Cells in Pediatric, Adolescent and Young Adult Patients with Central Nervous System Tumors.

Tumors of the central nervous system (CNS) are the most common and lethal childhood malignancy. Detection of residual disease and longitudinal monitoring of treatment response in patients are challenging and rely on serial imaging. This current standard of care fails to detect microscopic disease or provide molecular characteristics of residual tumors. As such, there is dire need for minimally invasive liquid biopsy techniques. We have previously shown the high specificity of using cell surface vimentin (CSV) to identify circulating tumor cells (CTCs) from patients bearing various types of cancers. Here, we describe the first report of CTCs captured from peripheral blood samples in 58 pediatric CNS tumor patients. In this study, we used a CSV-coated cell capture chip, the Abnova CytoQuest automated CTC isolation system, to boost the CTC capture from pediatric patients with CNS tumors. We successfully isolated CTCs in six glioma patients using immunostaining of histone H3 lysine27-to-methionine (H3K27M) mutations which are highly expressed by this tumor. We show that CSV is a viable marker for CNS CTC isolation and that this is a feasible method for detecting microscopic disease. Larger-scale studies focusing on CTCs in pediatric CNS tumors to explore their diagnostic and prognostic value are warranted.

FGL2-targeting T cells exhibit antitumor effects on glioblastoma and recruit tumor-specific brain-resident memory T cells.

Although tissue-resident memory T (TRM) cells specific for previously encountered pathogens have been characterized, the induction and recruitment of brain TRM cells following immune therapy has not been observed in the context of glioblastoma. Here, we show that T cells expressing fibrinogen-like 2 (FGL2)-specific single-chain variable fragments (T-αFGL2) can induce tumor-specific CD8+ TRM cells that prevent glioblastoma recurrence. These CD8+ TRM cells display a highly expanded T cell receptor repertoire distinct from that found in peripheral tissue. When adoptively transferred to the brains of either immunocompetent or T cell-deficient naïve mice, these CD8+ TRM cells reject glioma cells. Mechanistically, T-αFGL2 cell treatment increased the number of CD69+CD8+ brain-resident memory T cells in tumor-bearing mice via a CXCL9/10 and CXCR3 chemokine axis. These findings suggest that tumor-specific brain-resident CD8+ TRM cells may have promising implications for the prevention of brain tumor recurrence.

Competitive DNA transfection formulation via electroporation for human adipose stem cells and mesenchymal stem cells.

BACKGROUND: Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use. RESULTS: We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells. CONCLUSIONS: Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.

Discovery of a linear peptide for improving tumor targeting of gene products and treatment of distal tumors by IL-12 gene therapy.

Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.

A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions.

Receptor-ligand binding has been analyzed at the protein level using isothermal titration calorimetry and surface plasmon resonance and at the cellular level using interaction-associated downstream gene induction/suppression. However, no currently available technique can characterize this interaction directly through visualization. In addition, all available assays require a large pool of cells; no assay capable of analyzing receptor-ligand interactions at the single-cell level is publicly available. Here, we describe a new microfluidic chip-based technique for analyzing and visualizing these interactions at the single-cell level. First, a protein is immobilized on a glass slide and a low-flow-rate pump is used to isolate cells that express receptors that bind to the immobilized ligand. Specifically, we demonstrate the efficacy of this technique by immobilizing biotin-conjugated FGL2 on an avidin-coated slide chip and passing a mixture of GFP-labeled wild-type T cells and RFP-labeled FcγRIIB-knockout T cells through the chip. Using automated scanning and counting, we found a large number of GFP+ T cells with binding activity but significantly fewer RFP+ FcγRIIB-knockout T cells. We further isolated T cells expressing a membrane-anchored, tumor-targeted IL-12 based on the receptor's affinity to vimentin to confirm the versatility of our technique. This protocol allows researchers to isolate receptor-expressing cells in about 4 hours for further downstream processing.

Membrane-Anchored and Tumor-Targeted IL12 (attIL12)-PBMC Therapy for Osteosarcoma.

PURPOSE: Chimeric antigen receptor (CAR) T-cell therapy has shown great promise for treating hematologic malignancies but requires a long duration of T-cell expansion, is associated with severe toxicity, and has limited efficacy for treating solid tumors. We designed experiments to address those challenges. EXPERIMENTAL DESIGN: We generated a cell membrane-anchored and tumor-targeted IL12 (attIL12) to arm peripheral blood mononuclear cells (PBMC) instead of T cells to omit the expansion phase for required CAR T cells. RESULTS: This IL12-based attIL12-PBMC therapy showed significant antitumor efficacy in both heterogeneous osteosarcoma patient-derived xenograft tumors and metastatic osteosarcoma tumors with no observable toxic effects. Mechanistically, attIL12-PBMC treatment resulted in tumor-restricted antitumor cytokine release and accumulation of attIL12-PBMCs in tumors. It also induced terminal differentiation of osteosarcoma cells into bone-like cells to impede tumor growth. CONCLUSIONS: In summary, attIL12-PBMC therapy is safe and effective against osteosarcoma. Our goal is to move this treatment into a clinical trial. Owing to the convenience of the attIL12-PBMC production process, we believe it will be feasible.