The role of recruited bone-marrow derived mesenchymal stem cells in the establishment of the tumor stroma and a growth-supportive environment

The Mesenchymal Stem Cell (MSC) is a self-renewing multipotent progenitor originally found in the bone marrow. It has drawn strong interest from translational research because of the multipotency of MSCs, their immunosuppression, their intrinsic homing and their promotion of wound healing in human patients. Currently available methods rely on in vitro culture for the isolation and expansion of MSCs and most studies used these cells for their investigation. Fibroblasts constitute the connective tissue and support epithelial or hematopoietic cells. Although they have already been extensively investigated, it is not clear whether stem cells exist in vivo which specifically regenerate fibroblasts. MSCs are a potential candidate, because of their fibroblastic shape in vitro, their differentiation towards several mesodermal lineages and their supportive function. We aim to characterize the in vivo differentiation potential of MSCs toward fibroblasts in normal and tumor tissues. A new transplantation approach was developed in order to transfer labeled MSCs in the bone marrow and assess their recruitment and lineage contribution in vivo. Long-term engraftment was accomplished by isolating and transplanting these cells within one day. To this end, strategies for their purification, preconditioning of the host and transplantation methodologies were established during this thesis. We were able to enrich MSCs over 1000 fold by combining a new extraction protocol, a negative selection by MACS and a positive selection by FACS. Cloning of sorted MSCs was performed to confirm their multipotency at single cell level. GFP-expressing MSCs were able to engraft in host mice tolerant for GFP and their expansion in vivo was stimulated by block of GSK3 activity. Altogether, the chimeric MSC model was used to track the recruitment of bone-marrow derived MSCs into spontaneous breast tumors. The tumor microenvironment encompasses several non-tumorigenic cells interacting with cancer cells. Among them, cancer-associated fibroblasts are known to participate in carcinogenesis. Whilst this heterogeneous stromal population has been extensively investigated, their source(s) remain elusive. I now propose tumoral MSCs as one source of cancer-associated fibroblast, based on their ability to self-renew and reconstitute stromal heterogeneity upon serial transplantation. Finally, I introduced the new concept that the multipotency of MSCs is subjugated by cancer cells to meet their requirements for enhanced tumor progression and prepare for metastasis. I found MSCs present in the tumor microenvironment to differentiate into cancer-associated osteoblasts and produce hydroxyapatite-mineralization in vivo. Microcalcification is an accepted poor prognostic feature in breast cancer patients but its function and origin is not documented. I showed that hydroxyapatite was able to promote proliferation of tumor cells and increased intracellular calcium concentration. Lastly, cancer-associated osteoblast and hydroxyapatite were able to promote tumor growth and metastasis in vivo. To conclude, bone marrow derived MSCs are circulating stem cells that participate in the establishment of the pathogenic stroma in breast cancer. Owing to their multipotency, MSCs give rise to cancer-associated osteoblasts that induce pro-malignant mineralization in situ. Subsequently, cancer cells harbor increased intracellular calcium concentration, enhanced proliferation and metastasis. We foresee the development of new stroma-targeted therapies that prevent the mineralization of tumor tissue and reduce metastasis in breast cancer patients.

In vitro Fate Mapping of Single Hematopoietic Stem Cells

Aline Roch

The in vitro expansion of hematopoietic stem cells (HSC) for clinical applications is hampered by a rapid loss of HSC blood reconstitution capability in culture. While these rare cells can be stimulated to massively proliferate, cell divisions mostly result in differentiation caused by the lack of interactions with the native microenvironment, termed niche, in the bone marrow. Indeed, an increasing body of literature highlights the crucial role of instructive niche signals directing HSC fate in vivo. But how HSC fate, and in particular the delicate choice between self-renewal and differentiation divisions, is controlled by niche signaling cues remains unknown. Therefore, the goal of this thesis was to systematically characterize HSC fate decisions in vitro, and to utilize this knowledge to design artificial niches that maintain stemness. Differentiating HSCs first give rise to several transient populations of highly proliferative multipotent progenitors. Because reliable markers that distinguish HSCs from the earliest multipotent progenitors in vitro are lacking, it is currently not possible to discriminate between HSC self-renewal and commitment fate choices. The development and application of novel tools to probe HSC fate at the single cell level is therefore crucial, even more so because HSC populations are highly heterogeneous. In the first part of this thesis, a micro-engineered single cell analysis platform was employed to track in high-throughput the fate of individual HSCs by time-lapse microscopy. Single cell imaging revealed a surprising direct generation of megakaryocytes, cells that produce blood platelets, from phenotypic HSCs in the complete absence of a cell division. Megakaryocytic differentiation has previously been shown to occur very early in hematopoiesis and recent studies revealed the existence of a subpopulation of HSCs that is predetermined to undergo megakaryocytic differentiation. Our results suggest that some megakaryocyte progenitors may not be directly derived from HSCs but rather share the same cell surface marker repertoire such that they are indistinguishable from HSCs by state-of-the-art purification strategies. In order to discriminate between HSC self-renewal and commitment at single cell level, in the second part of this thesis gene expression signatures associated with the stem cell and multipotent progenitor cell states were established. Twelve differentially expressed genes marking the quiescent HSC state were identified, including four genes encoding cellcell interaction signals in the niche. Single cell multigene expression analysis performed on daughter cells, derived from single HSCs in serum-free culture, showed a rapid loss of the HSC identity with increasing number of cell divisions. In order to prevent such a dramatic loss of stemness in vitro, biomimetic microenvironments were engineered to display ligands of the newly identified niche components. Exposure of single HSCs to these artificial niches revealed a reduction in the mitotic activity of HSCs. Strikingly, in vivo transplantation of artificial niche-cultured HSCs resulted in long-term blood reconstitution of irradiated mice, demonstrating maintenance of functional HSC in vitro when exposed to critical cell-cell interaction signals found in native niches. Studies with invertebrates have shown that the pool of stem cells in vivo is controlled through asymmetric self-renewal divisions, resulting in two daughter cells with distinct fates, only one of which maintains stemness. Very little is known about asymmetric divisions of HSCs. Therefore, in the third part of this thesis, the symmetry of HSC divisions was systematically investigated in vitro. Using time-lapse imaging and single cell multigene expression analysis of paired daughter cells isolated by micromanipulation, approximately one third of all HSC was found to divide in an asymmetric fashion under serum-free culture conditions. Strikingly, paired daughter cells of cultured HSCs that were activated in vivo to exit their dormant state were found to divide mostly in an asymmetric fashion. These results shed light on the critical role of the in vivo microenvironment in specifying HSC fate and in particular asymmetric cell divisions. Altogether, this thesis demonstrates the power of single cell analyses to unravel stem cell fate decisions, and presents a novel approach to identify functional artificial niches to maintain HSCs in culture. This experimental paradigm should contribute to the development of better strategies to study and manipulate other stem cell types in culture. One exciting avenue is the expansion of human cord blood-derived HSCs, a cellular source that is particularly plagued by a lack of sufficient numbers for transplantation, with the aim of improving HSC therapies.

EPFL2014

Bioengineering approaches to emulate the stem cell niche

Sylke Höhnel

Stem cell therapies hold tremendous potential for tissue and organ regeneration. Yet, there remains significant need for better ex vivo culture and manipulation methods. On the one hand, many tissue-specific stem cells cannot be propagated without causing rapid deviations in cellular phenotype. On the other hand, outside of a developing organism, it remains very difficult to differentiate stem cells into mature functional cell types. Such protocols could help to build more effective and predictive pre-clinical models that would reduce or even replace animal studies. Manipulating a stem cell in vitro requires a thorough understanding of its microenvironment, or niche, and how niche factors influence stem cell fate. A customizable microarray platform was therefore developed to examine the interplay of biochemical and mechanical signals on stem cell behavior. This array was implemented to study combinations of 12 different proteins with varying substrate stiffness and their effect on the adipogenic differentiation of human mesenchymal stem cells (MSCs). These cells and their differentiated progenies are of high therapeutic relevance for skeletal tissue regeneration and have also been implied as niche-modulating neighbors of hematopoietic stem cells (HSCs) in the bone marrow (BM). Using this combinatorial approach, substrate stiffness was found to be the major determinant of the frequency of lipid accumulation in MSCs, while the lipid content was found to be strongly driven by the biochemical context. To further understand how niche cells together with stem cells self-organize to form a functional tissue, three-dimensional (3D) organ-mimicking structures from spheroids of pluri- and multi-potent have recently been developed. These systems, termed organoids, are expected to provide instrumental insights into tissue or organ development and they could be of great use for disease-specific drug screenings and to generate clinically transplantable tissues. However, current protocols for organoid generation remain highly inefficient and irreproducible. To perform the high-throughput aggregation of one or multiple cell types and to allow their long-term culture, a high-density array of U-shaped microwells was developed. This platform was used to initiate 3D co-cultures of the hematopoiesis-supportive cell line OP9 together with HSCs. To expand on the potential of this co-culture model to study more relevant primary BM cells, uniform clonal mesensphere cultures from human MSCs were established. However, restricted knowledge about the phenotypic identity of both niche and stem cell is a major limitation to initiate these co-cultures in vitro. Therefore, in this thesis a cell-targeting approach through covalent SNAP-tag-based cell-pairing is proposed. Specifically, SNAP-tag fusion proteins expressed on cell membranes were targeted to form covalent bonds with substrates that harbor the SNAP-tag substrate benzylguanine (BG). Immobilizing sufficient amounts of BG on cellular membranes is a mean to enable cell-cell pairing as demonstrated by the covalent aggregation of microbeads carrying SNAP-tag and BG at high concentrations. The artificial niche models developed in this study are broadly applicable to desired cellular systems. These approaches help to gain insight into the multifactorial composition and architectural organization of specific niches and to enable their reconstruction in vitro.

EPFL2015

Elucidating the role of lymphatic vessels within the tumor microenvironment of breast cancer

Ingrid Maria van Mier

Breast cancer is the most common cancer in women worldwide, with an estimated 1.7 million newly diagnosed cases in 2012. Despite the many clinical manifestations, metastatic disease is the main cause of death, and is responsible for 90% of all breast cancer-related deaths. Aggressive carcinomas of the breast preferentially spread via the lymphogenous route, and often co-opt and actively remodel the lymphatic vasculature in order to invade and metastasize to their draining lymph nodes (LNs) and, ultimately, to distant organs. Indeed, expression of vascular endothelial growth factor (VEGF)-C, the principal lymphangiogenic growth factor, by the tumor or recruited ancillary cells has been shown to correlate both with LN and distant metastasis, and shorter overall survival (OS), and LN status represents an important prognostic factor for patients diagnosed with breast cancer. Although traditionally considered as passive conduits that solely mediate tumor cell escape, more recent research aimed at discerning the interactions between the developing tumor and its associated lymphatic vessels has revealed that the lymphatic vasculature actively promotes lymphogenous metastasis of invasive tumor cells. Likewise, collaborative interactions between tumor cells and a multitude of other tissue-resident and recruited ancillary cells, which together constitute the tumor microenvironment, not only coercively contribute to tumor growth, progression and metastasis, but also suggest that interactions among primed stromal cells themselves may be important in the process of metastatic dissemination as well. Notably, expansion of tumor-associated lymphatic vessels in the tumor periphery leads to increased lymphatic drainage from the tumor to the tumor-draining LN, and interstitial flow-induced mechanical changes may alter tensional homeostasis within the tumor through stimulation of local fibroblasts that remodel and stiffen the extracellular matrix (ECM). Furthermore, VEGF-C-induced remodeling of the tumor-associated lymphatic vasculature has been shown to directly suppress the anti-tumor immune response, thereby promoting tolerance to the nascent tumor. Thus, to explore whether tumor-associated lymphatic vessels promote tumor metastasis not only via interactions with the incipient tumor, but also through cross-talk with the surrounding stromal cells, the different components of the tumor microenvironment, specifically tensional homeostasis and anti-tumor immunity, were evaluated upon modulation of lymphatic vessel growth in various experimental models of breast cancer. Accordingly, inhibition of VEGFR-3-mediated signaling upon administration of an antagonistic antibody in a genetically engineered preclinical mouse model specifically reduced the number of lymphatic endothelial cells (LECs) in the tumor, whereas conversely, orthotopic inoculation of a stable murine breast cancer cell line with enhanced expression of VEGF-C induced an expansion of the lymphatic endothelium. Although either experimental approach affected either regional or distant metastatic dissemination of the primary tumor to LN or the lungs, respectively, these effects did not seem to be mediated by interactions of the tumor-associated lymphatic vasculature with the tumor microenvironment in these particular experimental models.

EPFL2017