Reduction of product composition variability using pooled microbiome ecosystem therapy and consequence in two infectious murine models.

Growing evidence demonstrates the key role of the gut microbiota in human health and disease. The recent success of microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on its potential in conditions associated with gut dysbiosis, such as acute graft-versus-host disease, intestinal bowel diseases, neurodegenerative diseases, or even cancer. However, the difficulty in defining a "good" donor as well as the intrinsic variability of donor-derived products' taxonomic composition limits the translatability and reproducibility of these studies. Thus, the pooling of donors' feces has been proposed to homogenize product composition and achieve higher taxonomic richness and diversity. In this study, we compared the metagenomic profile of pooled products to corresponding single donor-derived products. We demonstrated that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria known to produce anti-inflammatory short chain fatty acids compared to single donor-derived products. We then evaluated pooled products' efficacy compared to corresponding single donor-derived products in Salmonella and C. difficile infectious mouse models. We were able to demonstrate that pooled products decreased pathogenicity by inducing a structural change in the intestinal microbiota composition. Single donor-derived product efficacy was variable, with some products failing to control disease progression. We further performed in vitro growth inhibition assays of two extremely drug-resistant bacteria, Enterococcus faecium vanA and Klebsiella pneumoniae oxa48, supporting the use of pooled microbiotherapies. Altogether, these results demonstrate that the heterogeneity of donor-derived products is corrected by pooled fecal microbiotherapies in several infectious preclinical models.IMPORTANCEGrowing evidence demonstrates the key role of the gut microbiota in human health and disease. Recent Food and Drug Administration approval of fecal microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on their potential to treat pathological conditions associated with gut dysbiosis. In this study, we combined metagenomic analysis with in vitro and in vivo studies to compare the efficacy of pooled microbiotherapy products to corresponding single donor-derived products. We demonstrate that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria compared to single donor-derived products. We further reveal that pooled products decreased Salmonella and Clostridioides difficile pathogenicity in mice, while single donor-derived product efficacy was variable, with some products failing to control disease progression. Altogether, these findings support the development of pooled microbiotherapies to overcome donor-dependent treatment efficacy.

L-asparaginase anti-tumor activity in pancreatic cancer is dependent on its glutaminase activity and resistance is mediated by glutamine synthetase.

l-Asparaginase is a cornerstone of acute lymphoblastic leukemia (ALL) therapy since lymphoblasts lack asparagine synthetase (ASNS) and rely on extracellular asparagine availability for survival. Resistance mechanisms are associated with increased ASNS expression in ALL. However, the association between ASNS and l-Asparaginase efficacy in solid tumors remains unclear, thus limiting clinical development. Interestingly, l-Asparaginase also has a glutaminase co-activity that is crucial in pancreatic cancer where KRAS mutations activate glutamine metabolism. By developing l-Asparaginase-resistant pancreatic cancer cells and using OMICS approaches, we identified glutamine synthetase (GS) as a marker of resistance to l-Asparaginase. GS is the only enzyme able to synthesize glutamine, and its expression also correlates with l-Asparaginase efficacy in 27 human cell lines from 11 cancer indications. Finally, we further demonstrated that GS inhibition prevents cancer cell adaptation to l-Asparaginase-induced glutamine starvation. These findings could pave the way to the development of promising drug combinations to overcome l-Asparaginase resistance.

Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process.

Red blood cells (RBCs) can act as carriers for therapeutic agents and can substantially improve the safety, pharmacokinetics, and pharmacodynamics of many drugs. Maintaining RBCs integrity and lifespan is important for the efficacy of RBCs as drug carrier. We investigated the impact of drug encapsulation by hypotonic dialysis on RBCs physiology and integrity. Several parameters were compared between processed RBCs loaded with l-asparaginase ("eryaspase"), processed RBCs without drug and non-processed RBCs. Processed RBCs were less hydrated and displayed a reduction of intracellular content. We observed a change in the metabolomic but not in the proteomic profile of processed RBCs. Encapsulation process caused moderate morphological changes and was accompanied by an increase of RBCs-derived Extracellular Vesicles release. Despite a decrease in deformability, processed RBCs were not mechanically retained in a spleen-mimicking device and had increased surface-to-volume ratio and osmotic resistance. Processed RBCs half-life was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBCs prolonged its in vivo half-life compared to free forms. Our study demonstrated that encapsulation by hypotonic dialysis may affect certain characteristics of RBCs but does not significantly affect the in vivo longevity of RBCs or their drug carrier function.

Lens-free microscopy for 3D + time acquisitions of 3D cell culture.

Thanks to a novel three-dimensional imaging platform based on lens-free microscopy, it is possible to perform multi-angle acquisitions and holographic reconstructions of 3D cell cultures directly into the incubator. Being able of reconstructing volumes as large as ~5 mm3 over a period of time covering several days, allows us to observe a broad range of migration strategies only present in 3D environment, whether it is single cell migration, collective migrations of cells and dispersal of cells. In addition we are able to distinguish new interesting phenomena, e.g. large-scale cell-to-matrix interactions (>1 mm), fusion of cell clusters into large aggregate (~10,000 µm2) and conversely, total dissociation of cell clusters into clumps of migrating cells. This work on a novel 3D + time lens-free microscopy technique thus expands the repertoire of phenomena that can be studied within 3D cell cultures.

Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions.

The spatiotemporal program of metazoan DNA replication is regulated during development and altered in cancers. We have generated novel OK-seq, Repli-seq and RNA-seq data to compare the DNA replication and gene expression programs of twelve cancer and non-cancer human cell types. Changes in replication fork directionality (RFD) determined by OK-seq are widespread but more frequent within GC-poor isochores and largely disconnected from transcription changes. Cancer cell RFD profiles cluster with non-cancer cells of similar developmental origin but not with different cancer types. Importantly, recurrent RFD changes are detected in specific tumour progression pathways. Using a model for establishment and early progression of chronic myeloid leukemia (CML), we identify 1027 replication initiation zones (IZs) that progressively change efficiency during long-term expression of the BCR-ABL1 oncogene, being twice more often downregulated than upregulated. Prolonged expression of BCR-ABL1 results in targeting of new IZs and accentuation of previous efficiency changes. Targeted IZs are predominantly located in GC-poor, late replicating gene deserts and frequently silenced in late CML. Prolonged expression of BCR-ABL1 results in massive deletion of GC-poor, late replicating DNA sequences enriched in origin silencing events. We conclude that BCR-ABL1 expression progressively affects replication and stability of GC-poor, late-replicating regions during CML progression.

Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens.

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.

Immature CML cells implement a BMP autocrine loop to escape TKI treatment.

The BCR-ABL specific tyrosine kinase inhibitors (TKI) changed the outcome of chronic myeloid leukemia (CML), turning a life-threatening disease into a chronic illness. However, TKI are not yet curative, because most patients retain leukemic stem cells (LSC) and their progenitors in bone marrow and relapse following treatment cessation. At diagnosis, deregulation of the bone morphogenetic protein (BMP) pathway is involved in LSC and progenitor expansion. Here, we report that BMP pathway alterations persist in TKI-resistant patients. In comparison with patients in complete cytogenetic remission, TKI-resistant LSC and progenitors display high levels of BMPR1b expression and alterations of its cellular localization. In vitro treatment of immature chronic phase CML cells with TKI alone, or in combination with interferon-α, results in the preferential survival of BMPR1b+ cells. We demonstrated persistent and increasing BMP4 production by patients' mesenchymal cells with resistance. Patient follow-up revealed an increase of BMPR1b expression and in BMP4 expression in LSC from TKI-resistant patients in comparison with diagnosis, while remaining unchanged in sensitive patients. Both leukemic and nonleukemic cells exhibit higher BMP4 levels in the bone marrow of TKI-resistant patients. Exposure to BMP2/BMP4 does not alter BCR-ABL transcript expression but is accompanied by the overexpression of TWIST-1, a transcription factor highly expressed in resistant LSC. By modulating BMP4 or BMPR1b expression, we show that these elements are involved in TKI resistance. In summary, we reveal that persistence of BMP alterations and existence of an autocrine loop promote CML-primitive cells' TKI resistance.

Comparative study of fully three-dimensional reconstruction algorithms for lens-free microscopy.

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multiangle acquisitions on 3D cell cultures embedded in extracellular matrices. Lens-free microscopy acquisitions present some inherent issues such as the lack of phase information on the sensor plane and a limited angular coverage. We developed and compared three different algorithms based on the Fourier diffraction theorem to obtain fully 3D reconstructions. These algorithms present an increasing complexity associated with a better reconstruction quality. Two of them are based on a regularized inverse problem approach. To compare the reconstruction methods in terms of artefact reduction, signal-to-noise ratio, and computation time, we tested them on two experimental datasets: an endothelial cell culture and a prostate cell culture grown in a 3D extracellular matrix with large reconstructed volumes up to ∼5 mm3 with a resolution sufficient to resolve isolated single cells. The lens-free reconstructions compare well with standard microscopy.

Deciphering the internal complexity of living cells with quantitative phase microscopy: a multiscale approach.

The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

Primitive CML cell expansion relies on abnormal levels of BMPs provided by the niche and on BMPRIb overexpression.

Leukemic stem cells in chronic phase chronic myelogenous leukemia (CP-CML) are responsible for disease persistence and eventual drug resistance, most likely because they survive, expand, and are sustained through interactions with their microenvironment. Bone morphogenetic proteins 2 (BMP2) and 4 (BMP4) regulate the fate and proliferation of normal hematopoietic stem cells, as well as interactions with their niche. We show here that the intrinsic expression of members of the BMP response pathway are deregulated in CML cells with differences exhibited in mature (CD34(-)) and immature (CD34(+)) compartments. These changes are accompanied by altered functional responses of primitive leukemic cells to BMP2 and BMP4 and strong increases in soluble BMP2 and BMP4 in the CML bone marrow. Using primary cells and a cell line mimicking CP-CML, we found that myeloid progenitor expansion is driven by the exposure of immature cells overexpressing BMP receptor Ib to BMP2 and BMP4. In summary, we demonstrate that deregulation of intracellular BMP signaling in primary CP-CML samples corrupts and amplifies their response to exogenous BMP2 and BMP4, which are abnormally abundant within the tumor microenvironment. These results provide new insights with regard to leukemic stem cell biology and suggest possibilities for the development of novel therapeutic tools specifically targeting the CML niche.

[BMP and cancer: the Yin and Yang of stem cells]. / BMP et cancer: le Yin et le Yang des cellules souches.

In a normal context, bone morphogenetic proteins (BMPs), members of the TGFß superfamily, are key players in adult stem cell biology. They are involved in the control of the overall functional and phenotypic properties of the stem cell population (self-renewal, proliferation, differentiation, apoptosis, quiescence, etc.). They can act directly on the stem cell or through its microenvironment, contributing to the tight balance of this system. In the tumorigenic context, alterations of the BMP signalling are involved in the deregulation of the interaction between stem cells and their microenvironment and, as such, participate to the different steps of the transformation process.