Lung Allograft Microbiome Association with Gastroesophageal Reflux, Inflammation, and Allograft Dysfunction.

Rationale: It remains unclear how gastroesophageal reflux disease (GERD) affects allograft microbial community composition in lung transplant recipients and its impact on lung allograft inflammation and function. Objectives: Our objective was to compare the allograft microbiota in lung transplant recipients with or without clinically diagnosed GERD in the first year after transplant and assess associations between GERD, allograft microbiota, inflammation, and acute and chronic lung allograft dysfunction (ALAD and CLAD). Methods: A total of 268 BAL samples were collected from 75 lung transplant recipients at a single transplant center every 3 months after transplant for 1 year. Ten transplant recipients from a separate transplant center provided samples before and after antireflux Nissen fundoplication surgery. Microbial community composition and density were measured using 16S ribosomal RNA gene sequencing and quantitative polymerase chain reaction, respectively, and inflammatory markers and bile acids were quantified. Measurements and Main Results: We observed a range of allograft community composition with three discernible types (labeled community state types [CSTs] 1-3). Transplant recipients with GERD were more likely to have CST1, characterized by high bacterial density and relative abundance of the oropharyngeal colonizing genera Prevotella and Veillonella. GERD was associated with more frequent transitions to CST1. CST1 was associated with lower inflammatory cytokine concentrations than pathogen-dominated CST3 across the range of microbial densities observed. Cox proportional hazard models revealed associations between CST3 and the development of ALAD/CLAD. Nissen fundoplication decreased bacterial load and proinflammatory cytokines. Conclusions: GERD was associated with a high bacterial density, Prevotella- and Veillonella-dominated CST1. CST3, but not CST1 or GERD, was associated with inflammation and early development of ALAD and CLAD. Nissen fundoplication was associated with a reduction in microbial density in BAL fluid samples, especially the CST1-specific genus, Prevotella.

Therapeutic interventions and mechanisms associated with gut microbiota-mediated modulation of immune checkpoint inhibitor responses.

The link between the gut microbiome and responsiveness to immune checkpoint inhibitor (ICI) therapy is now well established. New therapeutic opportunities exploiting this relationship are being developed with the goal of augmenting ICI efficacy. In this review, we summarize the foundational research establishing these interactions and discuss the mechanisms and novel therapeutic options associated with this gut microbiome-ICI connection.

Variable impact of three different antiangiogenic drugs alone or in combination with chemotherapy on multiple bone marrow-derived cell populations involved in angiogenesis and immunity.

In contrast to VEGF pathway-targeting antibodies, antiangiogenic tyrosine kinase inhibitors (TKIs) have failed to meet primary endpoints in almost all phase III clinical trials when combined with conventional chemotherapy. One exception is the combination of nintedanib and docetaxel as a second-line therapy for rapidly progressing advanced NSCLC. In addition to increased toxicity caused by this type of combination, thus necessitating drug dose reductions or treatment breaks, such phase III trial failures may also be related to the differential impact of host-mediated responses involving mobilization and tumor infiltration of bone marrow-derived cell populations (BMDCs), comprising both pro-angiogenic as well as immune effector cells. Herein, we evaluated two different antiangiogenic TKIs (sunitinib or nintedanib) and a VEGFR-2 antibody (DC101) either alone or combined with maximum tolerated dose paclitaxel for their differential impact on the BMDC host response, evaluating four different cell types. TKIs (in particular sunitinib) induced myelosuppression similar to paclitaxel, whereas DC101 had no such effect. Sunitinib also significantly decreased the number of tumor-infiltrating CD8 + T and B cells, MDSCs, and macrophages. In contrast, the effect of nintedanib on these BMDC populations was less marked, behaving closer to the VEGFR-2 antibody effects than sunitinib. The results raise the possibility that differences observed between antiangiogenic antibodies and TKIs in increasing chemotherapy efficacy could be related, at least in part, to differential effects on cells associated with local immunity within the tumor microenvironment.

Therapeutic impact of Nintedanib with paclitaxel and/or a PD-L1 antibody in preclinical models of orthotopic primary or metastatic triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive malignancy with poor prognosis, in part because of the current lack of any approved molecularly targeted therapy. We evaluated various combinations of three different drugs: nintedanib, an antiangiogenic TKI targeting VEGF receptors, paclitaxel (PTX), or a PD-L1 antibody, using models of orthotopic primary or advanced metastatic TNBC involving a metastatic variant of the MDA-MB-231 human cell line (called LM2-4) in SCID mice and two mouse lines (EMT-6 and a drug-resistant variant, EMT-6/CDDP) in immunocompetent mice. These drugs were selected based on the following: PTX is approved for TNBC; nintedanib combined with docetaxel has shown phase III clinical trial success, albeit in NSCLC; VEGF can act as local immunosuppressive factor; and PD-L1 antibody plus taxane therapy was recently reported to have encouraging phase III trial benefit in TNBC. METHODS: Statistical analyses were performed with ANOVA followed by Tukey's Multiple Comparison Test or with Kruskal-Wallis test followed by Dunn's Multiple Comparison Test. Survival curves were analyzed using a Log-rank (Mantel Cox) test. Differences were considered statistically significant when p values were < 0.05. RESULTS: Toxicity analyses showed that nintedanib is well tolerated when administered 5-days ON 2-days OFF; PTX toxicity differed in mice, varied with cell lines used and may have influenced median survival in the metastatic EMT6/CDDP model; while toxicity of PD-L1 therapy depended on the cell lines and treatment settings tested. In the LM2-4 system, combining nintedanib with PTX enhanced overall antitumor efficacy in both primary and metastatic treatment settings. In immunocompetent mice, combining nintedanib or PTX with the PD-L1 antibody improved overall antitumor efficacy. Using the advanced metastatic EMT-6/CDDP model, optimal efficacy results were obtained using the triple combination. CONCLUSIONS: These results suggest circumstances where nintedanib plus PTX may be potentially effective in treating TNBC, and nintedanib with PTX may improve PD-L1 therapy of metastatic TNBC.

Preclinical impact of high dose intermittent antiangiogenic tyrosine kinase inhibitor pazopanib in intrinsically resistant tumor models.

Antiangiogenic tyrosine kinase inhibitors (TKIs) target vascular endothelial growth factor receptors and other receptor tyrosine kinases. As a result of toxicity, the clinical failures or the modest benefits associated with antiangiogenic TKI therapy may be related in some cases to suboptimal drug dosing and scheduling, thereby facilitating resistance. Most antiangiogenic TKIs, including pazopanib, are administered on a continuous daily basis. Here, instead, we evaluated the impact of increasing the dose and administering the drug intermittently. The rationale is that using such protocols, antitumor efficacy could be enhanced by direct tumor cell targeting effects in addition to inhibiting tumor angiogenesis. To test this, we employed two human tumor xenograft models, both of which manifest intrinsic resistance to pazopanib when it is administered continuously: the VHL-wildtype SN12-PM6-1 renal cell carcinoma (RCC) and the metastatic MDA-MB-231/LM2-4 variant breast cancer cell line, when treated as distant metastases. We evaluated four different doses and schedules of pazopanib in the context of primary tumors and advanced metastatic disease, in both models. The RCC model was not converted to drug sensitivity using the intermittent protocol. Using these protocols did not enhance the efficacy when treating primary LM2-4 tumors. However, one of the high-dose intermittent pazopanib protocols increased median survival when treating advanced metastatic disease. In conclusion, these results overall suggest that primary tumors showing sensitivity to continuous pazopanib treatment may predict response to this drug when given at high doses intermittently in the context of advanced metastatic disease, that are otherwise resistant to the conventional protocol.

Pore size is a critical parameter for obtaining sustained protein release from electrochemically synthesized mesoporous silicon microparticles.

Mesoporous silicon has become a material of high interest for drug delivery due to its outstanding internal surface area and inherent biodegradability. We have previously reported the preparation of mesoporous silicon microparticles (MS-MPs) synthesized by an advantageous electrochemical method, and showed that due to their inner structure they can adsorb proteins in amounts exceeding the mass of the carrier itself. Protein release from these MS-MPs showed low burst effect and fast delivery kinetics with complete release in a few hours. In this work, we explored if tailoring the size of the inner pores of the particles would retard the protein release process. To address this hypothesis, three new MS-MPs prototypes were prepared by electrochemical synthesis, and the resulting carriers were characterized for morphology, particle size, and pore structure. All MS-MP prototypes had 90 µm mean particle size, but depending on the current density applied for synthesis, pore size changed between 5 and 13 nm. The model protein α-chymotrypsinogen was loaded into MS-MPs by adsorption and solvent evaporation. In the subsequent release experiments, no burst release of the protein was detected for any prototype. However, prototypes with larger pores (>10 nm) reached 100% release in 24-48 h, whereas prototypes with small mesopores (<6 nm) still retained most of their cargo after 96 h. MS-MPs with ∼6 nm pores were loaded with the osteogenic factor BMP7, and sustained release of this protein for up to two weeks was achieved. In conclusion, our results confirm that tailoring pore size can modify protein release from MS-MPs, and that prototypes with potential therapeutic utility for regional delivery of osteogenic factors can be prepared by convenient techniques.

Implantable controlled release devices for BMP-7 delivery and suppression of glioblastoma initiating cells.

Designing therapeutic devices capable of manipulating glioblastoma initiating cells (GICs) is critical to stop tumor recurrence and its associated mortality. Previous studies have indicated that bone morphogenetic protein-7 (BMP-7) acts as an endogenous suppressor of GICs, and thus, it could become a treatment for this cancer. In this work, we engineer an implantable microsphere system optimized for the controlled release of BMP-7 as a bioinspired therapeutic device against GICs. This microsphere delivery system is based on the formation of a heparin-BMP-7 nanocomplex, first coated with Tetronic(®) and further entrapped in a biodegradable polyester matrix. The obtained microspheres can efficiently encapsulate BMP-7, and release it in a controlled manner with minimum burst effect for over two months while maintaining protein bioactivity. Released BMP-7 showed a remarkable capacity to stop tumor formation in a GICs cell culture model, an effect that could be mediated by forced reprogramming of tumorigenic cells towards a non-tumorigenic astroglial lineage.