Transcriptome sequencing and multi-plex imaging of prostate cancer microenvironment reveals a dominant role for monocytic cells in progression.

BACKGROUND: Prostate cancer is caused by genomic aberrations in normal epithelial cells, however clinical translation of findings from analyses of cancer cells alone has been very limited. A deeper understanding of the tumour microenvironment is needed to identify the key drivers of disease progression and reveal novel therapeutic opportunities. RESULTS: In this study, the experimental enrichment of selected cell-types, the development of a Bayesian inference model for continuous differential transcript abundance, and multiplex immunohistochemistry permitted us to define the transcriptional landscape of the prostate cancer microenvironment along the disease progression axis. An important role of monocytes and macrophages in prostate cancer progression and disease recurrence was uncovered, supported by both transcriptional landscape findings and by differential tissue composition analyses. These findings were corroborated and validated by spatial analyses at the single-cell level using multiplex immunohistochemistry. CONCLUSIONS: This study advances our knowledge concerning the role of monocyte-derived recruitment in primary prostate cancer, and supports their key role in disease progression, patient survival and prostate microenvironment immune modulation.

Mesenchymal stem cells from cortical bone demonstrate increased clonal incidence, potency, and developmental capacity compared to their bone marrow-derived counterparts.

In this study, we show that matrix dense cortical bone is the more potent compartment of bone than bone marrow as a stromal source for mesenchymal stem cells as isolated from adult rats. Lineage-depleted cortical bone-mesenchymal stem cells demonstrated >150-fold enrichment of colony forming unit-fibroblasts per cell incidence. compared to lineage-depleted bone marrow-mesenchymal stem cells, corresponding to a 70-fold increase in absolute recovered colony forming unit-fibroblasts. The composite phenotype Lin(-)/CD45(-)/CD31(-)/VLA-1(+)/Thy-1(+) enriched for clonogenic mesenchymal stem cells solely from cortical bone-derived cells from which 70% of clones spontaneously differentiated into all lineages of bone, cartilage, and adipose. Both populations generated vascularized bone tissue within subcutaneous implanted collagen scaffolds; however, cortical bone-derived cells formed significantly more osteoid than bone marrow counterparts, quantified by histology. The data demonstrate that our isolation protocol identifies and validates mesenchymal stem cells with superior clonal, proliferative, and developmental potential from cortical bone compared to the bone marrow niche although marrow persists as the typical source for mesenchymal stem cells both in the literature and current pre-clinical therapies.

Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation.

Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-ß1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM.

Multi-composite bioactive osteogenic sponges featuring mesenchymal stem cells, platelet-rich plasma, nanoporous silicon enclosures, and Peptide amphiphiles for rapid bone regeneration.

A novel bioactive sponge was created with a composite of type I collagen sponges or porous poly(e-caprolactone) (PCL) scaffolds, platelet-rich plasma (PRP), BMP2-loaded nanoporous silicon enclosure (NSE) microparticles, mineralizing peptide amphiphiles (PA), and mesenchymal stem cells (MSC). Primary MSC from cortical bone (CB)  tissue proved to form more and larger colony units, as well as produce more mineral matrix under osteogenic differentiation, than MSC from bone marrow (BM). Coating pre-treatments were optimized for maximum cell adhesion and mineralization, while a PRP-based gel carrier was created to efficiently deliver and retain MSC and  microparticles within a porous scaffold while simultaneously promoting cell recruitment, proliferation, and angiogenesis. Components and composite sponges were evaluated for osteogenic differentiation in vitro. Osteogenic sponges were loaded with MSC, PRP, PA, and NSE and implanted subcutaneously in rats to evaluate the formation of bone tissue and angiogenesis in vivo. It was found that the combination of a collagen sponge with CB MSC, PRP, PA, and the BMP2-releasing NSE formed the most bone and was most vascularized by four weeks compared to analogous composites featuring BM MSC or PCL or lacking PRP, PA, and NSE. This study indicates that CB MSC should be considered as an alternative to marrow as a source of stem cells, while the PRP-PA cell and microparticle delivery system may be utilized for diverse tissue engineering applications.

A role for pericytes as microenvironmental regulators of human skin tissue regeneration.

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin alpha5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.