Decellularized Colorectal Cancer Matrices as Bioactive Scaffolds for Studying Tumor-Stroma Interactions

More than a physical structure providing support to tissues, the extracellular matrix (ECM) is a complex and dynamic network of macromolecules that modulates the behavior of both cancer cells and associated stromal cells of the tumor microenvironment (TME). Over the last few years, several efforts have been made to develop new models that accurately mimic the interconnections within the TME and specifically the biomechanical and biomolecular complexity of the tumor ECM. Particularly in colorectal cancer, the ECM is highly remodeled and disorganized and constitutes a key component that affects cancer hallmarks, such as cell differentiation, proliferation, angiogenesis, invasion and metastasis. Therefore, several scaffolds produced from natural and/or synthetic polymers and ceramics have been used in 3D biomimetic strategies for colorectal cancer research. Nevertheless, decellularized ECM from colorectal tumors is a unique model that offers the maintenance of native ECM architecture and molecular composition. This review will focus on innovative and advanced 3D-based models of decellularized ECM as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro 2D and in vivo models. Our aim is to highlight the need for strategies that accurately mimic the TME for precision medicine and for studying the pathophysiology of the disease.

Identification, Characterization, and Expression Analysis of Spondin-Like and Fasciclin-Like Genes in Neopyropia yezoensis, A Marine Red Alga

Extracellular matrix (ECM) proteins play crucial roles in the regulation of cell proliferation and differentiation. We identified homologous genes encoding ECM proteins that are known to associate with integrins in animal cells in red macroalga Neopyropia yezoensis. Four genes encoding spondin domain-containing proteins (NySPLs) and eight genes encoding fasciclin domain-containing proteins (NyFALs) from N. yezoensis were selected for bioinformatics and expression analysis in order to obtain insights into the roles of ECM proteins for the life cycle. NySPLs had eight β-strands with two contiguous α-helices, which were similar to those of the F-spondin domain of animals. NyFALs had conserved H1 and H2 motifs and a YH motif between the H1 and H2 regions. Quantitative reverse transcription polymerase chain reaction showed that NySPL1–3 and NyFAL8 transcripts were highly accumulated in mature gametophytes that formed the spermatia. Furthermore, expressions of all NySPLs were upregulated in response to the ethylene precursor 1-aminocylopropane-1-carboxylic acid that induces gametogenesis. NyFAL1, 4 were highly expressed in sporophytes, whereas NyFAL2, 3, 5, 6, and 7 were overexpressed in gametophytes, especially at the vegetative stage. These findings facilitate future research on ECM architecture in the unique life cycles of red macroalgae.

Interindividual heterogeneity affects the outcome of human cardiac tissue decellularization

The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold’s intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

P–699 Multi-scale study of the architecture, topography and mechanics of the human ovary from prepuberty to menopause: a blueprint for next-generation bioengineering and diagnosis

Study question Does the ovarian ECM have a precise and unique biophysical phenotype, specific to each age, from prepuberty to menopause? Summary answer Differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. What is known already Ovarian engineering has recently emerged to respond to patient needs and offer reliable models for basic research. It has relied on synthetic and natural biomaterials and microfluidics. However, these techniques were designed based on knowledge acquired from 2D cell culture and animal models. Our lack of information on the human ovary hampers our ability to mimic the main features of this organ, for clinical applications. The complex composition and hierarchical structure of its ECM complicates the design of truly biomimetic constructs, notably: fiber morphology, interstitial and perifollicular fiber orientation, porosity, topography, and viscoelasticity, which all play a role in mechanotransduction. Study design, size, duration Ovarian biopsies were taken from prepubertal (mean age [±SD]=7±3 years, n = 21), reproductive age (mean age [±SD]=27±5, n = 26 ) and menopausal (mean age [±SD]=61±6 years, n = 29) patients after obtaining their informed consent. All participating adult subjects were undergoing laparoscopic surgery for benign gynecological diseases not affecting the ovaries. Prepubertal tissue was derived from young cancer patients scheduled for ovarian cortex cryopreservation as a fertility preservation strategy, before being subjected to acute gonadotoxic cancer treatments. Participants/materials, setting, methods All samples were cryopreserved by slow freezing and kept frozen until the day of their analysis. Tissues provided from the same patients (n = 5 per age group) were investigated by scanning electron microscopy (SEM) (fiber, pore and topography analyses) and atomic force microscopy (AFM). A larger number of paraffin-fixed biopsies (prepubertal, n = 16, reproductive-age, n = 21, and menopausal, n = 24) obtained from the biobank of St-Luc’s Hospital were used to conduct computed fiber orientation analysis. Main results and the role of chance Our results revealed a unique ECM architecture at reproductive age, where fibers of intermediate diameter are assembled into thickest bundles compared to prepubertal and menopausal tissues(p < 0.0001). Indeed, during prepuberty the bundles assemble into a tight network with high number of small pores while reproductive-age ovary gain more porosity(p < 0.0001). However, at menopause tissue pore number and area change significantly(p < 0.001). These pore geometry and distribution changes contribute to diffusion and access of key molecules to/from cells, which can be translated into changes in permeability and molecule selectivity with age. Fiber directionality around follicle borders at preantral stages revealed that before and after puberty, secondary follicles appear to modify their microenvironment arrangement locally compared to follicles at earlier stages of development (p < 0.01), by reorienting the majority of collagen fibers below 50°.This could indicate that follicles at this stage require higher fiber contact and adhesion signaling to complete their development and maturation towards ovulation. AFM evidenced a relatively rigid ovarian tissue at prepuberty, softening significantly at reproductive age, then stiffening considerably upon menopause. These differences(p < 0.01) are not only structure-dependent, but also related to biochemical differences in ECM composition, as previously demonstrated in our follow-up of variations in elastic matrisome components from prepuberty to menopause. Limitations, reasons for caution The samples represent single time points from each age group which could present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. Wider implications of the findings: Our study provides the first conclusive proof of a link between ECM biophysics and fertility by comparing different stages of ovarian transformation related to a woman’s reproductive life, which will oriente new strategies for infertility prognoses based on ECM biophysics and may become a blueprint for designing functional engineered ovaries. Trial registration number Not applicable

Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing 5 preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase (LOX) was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

Hemicentin-1 is An Essential Extracellular Matrix Component of the Dermal-Epidermal and Myotendinous Junctions

The extracellular matrix (ECM) architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (>600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal-epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1-/-mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1-/-mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.

Non-canonical Wnt/Ror2 signaling status regulates cell-matrix crosstalk to prompt directional tumor cell invasion and dissemination in breast cancer

Cancer deaths largely result from metastasis, the spread of cancer from the primary tumor to distant organs. Initial steps of metastasis require that tumor cells invade into the surrounding tissue and gain access to blood or lymphatic vessels. Such invasion is reliant on a balance of cell-cell and cell-matrix cues within the microenvironment of the tumor, yet factors regulating such interactions for invading tumor cells remain elusive in the context of cancer. We demonstrate that the noncanonical Wnt receptor, Ror2, in mammary tumor models of Tripe Negative Breast Cancer, regulates the composition and remodeling of the tumor stroma, where Ror2-depletion prompts directional tumor cell invasion and coordinated ECM production at the leading edge of tumor cell movement. By RNA sequencing, we discovered that tumor organoids specifically harbor actin cytoskeleton, cell adhesion, and collagen cross-linking gene expression programs when Wnt/Ror2 signaling is impaired. Interestingly, Ror2 depletion resulted in the downregulation of E-cadherin in tumor organoids, particularly at invading tumor cell protrusions within the surrounding ECM. Spatially, we identified the upregulation and redistribution of integrin receptors, particularly integrin-α5 in Ror2-deficient tumor organoids, accompanied by the simultaneous production of a provisional Fibronectin matrix, a requisite component of the ECM, ligand for integrin α5, and mediator of collagen assembly and organization. Along with altered ECM architecture, Ror2 loss reshaped the topology of integrin and FAK activation within primary tumors, suggesting an important physiological function for Ror2 in shaping both signaling and ECM architecture during tumor progression. Blocking either integrin or FAK, a downstream mediator of integrin-mediated signal transduction, abrogated the enhanced migration observed upon Ror2 loss. These results suggest that Ror2 status within a tumor can significantly impact adhesive vs. migratory states in breast cancer and provide a novel mechanism where Wnt/Ror2 shapes not only tumor cell composition, but also reciprocal cell-ECM interactions prompting directional and collaborative tumor cell transit during cancer progression.

Macrophage morphological plasticity and migration is Rac signalling and MMP9 dependant

In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilise haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation.

3D Scaffolds to Model the Hematopoietic Stem Cell Niche: Applications and Perspectives

Hematopoietic stem cells (HSC) are responsible for the production of blood and immune cells during life. HSC fate decisions are dependent on signals from specialized microenvironments in the bone marrow, termed niches. The HSC niche is a tridimensional environment that comprises cellular, chemical, and physical elements. Introductorily, we will revise the current knowledge of some relevant elements of the niche. Despite the importance of the niche in HSC function, most experimental approaches to study human HSCs use bidimensional models. Probably, this contributes to the failure in translating many in vitro findings into a clinical setting. Recreating the complexity of the bone marrow microenvironment in vitro would provide a powerful tool to achieve in vitro production of HSCs for transplantation, develop more effective therapies for hematologic malignancies and provide deeper insight into the HSC niche. We previously demonstrated that an optimized decellularization method can preserve with striking detail the ECM architecture of the bone marrow niche and support HSC culture. We will discuss the potential of this decellularized scaffold as HSC niche model. Besides decellularized scaffolds, several other methods have been reported to mimic some characteristics of the HSC niche. In this review, we will examine these models and their applications, advantages, and limitations.

Macrophage Plasticity is Rac Signalling and MMP9 Dependant

In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilisation haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation.