Leveraging mechanobiology and biophysical cues in lung organoids for studying lung development and disease

Lung organoids have emerged as powerful tools for studying lung distal diseases by recapitulating the cellular diversity and microenvironment of the lung tissue. This review article highlights the advancements in leveraging mechanobiology and biophysical cues in lung organoid engineering to improve their physiological relevance and disease modelling capabilities. We discuss the role of mechanobiology in lung development and homeostasis, as well as the integration of biophysical cues in the design and culture of lung organoids. Furthermore, we explore how these advancements have contributed to the understanding of lung distal diseases pathogenesis. We also discuss the challenges and future directions in harnessing mechanobiology and biophysical cues in lung organoid research. This review showcases the potential of lung organoids as a platform to investigate the underappreciated impacts of biophysical and biomechanical properties in enhancing lung organoids complexity and functionality, and ultimately provide new insight into embryonic lung development and pulmonary distal diseases pathogenesis

Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers.

In shade-intolerant plants such as Arabidopsis, a reduction in the red/far-red (R/FR) ratio, indicative of competition from other plants, triggers a suite of responses known as the shade avoidance syndrome (SAS). The phytochrome photoreceptors measure the R/FR ratio and control the SAS. The phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) are stabilized in the shade and are required for a full SAS, whereas the related bHLH factor HFR1 (long hypocotyl in FR light) is transcriptionally induced by shade and inhibits this response. Here we show that HFR1 interacts with PIF4 and PIF5 and limits their capacity to induce the expression of shade marker genes and to promote elongation growth. HFR1 directly inhibits these PIFs by forming non-DNA-binding heterodimers with PIF4 and PIF5. Our data indicate that PIF4 and PIF5 promote SAS by directly binding to G-boxes present in the promoter of shade marker genes, but their action is limited later in the shade when HFR1 accumulates and forms non-DNA-binding heterodimers. This negative feedback loop is important to limit the response of plants to shade

In vitro models of fetal lung development to enhance research into congenital lung diseases

PURPOSE: This paper aims to build upon previous work to definitively establish in vitro models of murine pseudoglandular stage lung development. These can be easily translated to human fetal lung samples to allow the investigation of lung development in physiologic and pathologic conditions. METHODS: Lungs were harvested from mouse embryos at E12.5 and cultured in three different settings, i.e., whole lung culture, mesenchyme-free epithelium culture, and organoid culture. For the whole lung culture, extracted lungs were embedded in Matrigel and incubated on permeable filters. Separately, distal epithelial tips were isolated by firstly removing mesothelial and mesenchymal cells, and then severing the tips from the airway tubes. These were then cultured either in branch-promoting or self-renewing conditions. RESULTS: Cultured whole lungs underwent branching morphogenesis similarly to native lungs. Real-time qPCR analysis demonstrated expression of key genes essential for lung bud formation. The culture condition for epithelial tips was optimized by testing different concentrations of FGF10 and CHIR99021 and evaluating branching formation. The epithelial rudiments in self-renewing conditions formed spherical 3D structures with homogeneous Sox9 expression. CONCLUSION: We report efficient protocols for ex vivo culture systems of pseudoglandular stage mouse embryonic lungs. These models can be applied to human samples and could be useful to paediatric surgeons to investigate normal lung development, understand the pathogenesis of congenital lung diseases, and explore novel therapeutic strategies

SHP-1 phosphatase activity counteracts increased T cell receptor affinity.

Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) < 1 μM) lead to drastic functional declines. Using human CD8(+) T cells engineered with TCRs of incremental affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 μM), those with supraphysiological affinity (K(d) = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity

Attracting cavities for docking. Replacing the rough energy landscape of the protein by a smooth attracting landscape.

Molecular docking is a computational approach for predicting the most probable position of ligands in the binding sites of macromolecules and constitutes the cornerstone of structure-based computer-aided drug design. Here, we present a new algorithm called Attracting Cavities that allows molecular docking to be performed by simple energy minimizations only. The approach consists in transiently replacing the rough potential energy hypersurface of the protein by a smooth attracting potential driving the ligands into protein cavities. The actual protein energy landscape is reintroduced in a second step to refine the ligand position. The scoring function of Attracting Cavities is based on the CHARMM force field and the FACTS solvation model. The approach was tested on the 85 experimental ligand-protein structures included in the Astex diverse set and achieved a success rate of 80% in reproducing the experimental binding mode starting from a completely randomized ligand conformer. The algorithm thus compares favorably with current state-of-the-art docking programs

On distributed strains in a CFA pile via DFOSs measurements and numerical analysis

Fibre Optic Sensors (FOSs) offer unprecedented possibilities for the monitoring of engineering structures, such as foundation systems. Notably, the type of FOSs known as Distributed Fibre Optic Sensors (DFOSs) has the capability of monitoring variations in the observed physical field, such as strain and temperature, with spatial continuity along the fibre. This paper presents and discusses the distributed strain measurements collected along a continuous flight auger (CFA) pile, belonging to the foundation raft of a new bridge subjected to an acceptance static load test. The monitoring was performed using a DFOS system, which works according to the optical frequency domain reflectometry (OFDR) method and provides a spatial resolution of 10 mm and a strain resolution of 1 \u3bc\u3b5. The in-situ monitoring results were used to calibrate a 3D Finite Element Model of the foundation system. The soil properties for the numerical model were also selected on the basis of a load test previously carried out on a similar pile at the same sit

CD73 expression and clinical significance in human metastatic melanoma.

CD73 is an ectoenzyme involved in the production of adenosine. It exerts immunosuppressive and protumoral roles and has emerged as a potential immuno-oncology target. CD73 expression was detected in TC in 54% of melanoma metastases, involving < 50% TC in the majority of the cases, with variable intensity. CD73 expression was significantly associated with a lower Breslow's depth of the primary lesion and was more frequent in patients having received prior non-surgical therapies. In an adjusted analysis, CD73 expression in TC (H-score > 37.5 or intensity > 1) significantly correlated to decreased overall survival (OS) from biopsy. Of the samples containing TIMC, 35% presented CD73+ TIMC. Highly infiltrated tumors were more likely to contain CD73+ TIMC. CD73 expression in TIMC (percentage ≥1%) significantly correlated with improved OS from biopsy. Immunohistochemistry detected CD73 expression in more than half of metastatic melanomas. While CD73 expression in TC significantly correlated with decreased OS, CD73 expression in TIMC significantly associated with improved OS. These results encourage the study of anti-CD73 therapies for metastatic melanoma patients. CD73 expression was assessed by immunohistochemistry in metastatic melanomas from 114 patients. Immunostainings were evaluated in tumor cells (TC) (percentage, intensity (1-3) and H-score) and in tumor-infiltrating mononuclear cells (TIMC) (percentage)

MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial.

BACKGROUND: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. METHODS: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. RESULTS: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7⁻ CD45RA⁺/⁻) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1⁻CD160⁻TIM3⁻LAG3⁻2B4⁺/⁻). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. CONCLUSIONS: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies

Durable response to palbociclib and letrozole in ovarian cancer with CDKN2A loss.

Alterations of the Retinoblastoma (Rb) pathway are frequent in ovarian cancer, typically resulting from CDKN2A down-regulation, CCNE1 amplification, CCND1/2 amplification, and RB1 loss. However, bi-allelic CDKN2A mutation or homozygous deletion is a very rare event, concerning less than 5% of patients.Initial trials with palbociclib in serous ovarian cancer have shown very modest benefit in unselected patient populations, thus underlining the need for a biomarker predicting response. We report the case of a heavily pre-treated patient with a serous ovarian tumor harboring a homozygous deletion of the CDKN2A gene that derived significant, prolonged clinical benefit from palbociclib, a CDK4/6 oral inhibitor, with letrozole. Treatment with palbociclib and letrozole started on February 2018, with an ongoing response after 12 months.In conclusion, homozygous CDKN2A deletion is rare and could be used to predict response to CDK4/6 inhibitors in association with other genomic features. We encourage further trials in this direction

Single cell analysis reveals similar functional competence of dominant and nondominant CD8 T-cell clonotypes.

Immune protection from infectious diseases and cancer is mediated by individual T cells of different clonal origin. Their functions are tightly regulated but not yet fully characterized. Understanding the contribution of each T cell will improve the prediction of immune protection based on laboratory assessment of T-cell responses. Here we developed techniques for simultaneous molecular and functional assessment of single CD8 T cells directly ex vivo. We studied two groups of patients with melanoma after vaccination with two closely related tumor antigenic peptides. Vaccination induced T cells with strong memory and effector functions, as found in virtually all T cells of the first patient group, and fractions of T cells in the second group. Interestingly, high functionality was not restricted to dominant clonotypes. Rather, dominant and nondominant clonotypes acquired equal functional competence. In parallel, this was also found for EBV- and CMV-specific T cells. Thus, the nondominant clonotypes may contribute similarly to immunity as their dominant counterparts