A living ex vivo platform for functional, personalized brain cancer diagnosis.

Functional precision medicine platforms are emerging as promising strategies to improve pre-clinical drug testing and guide clinical decisions. We have developed an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm that enable rapid engraftment, treatment, and analysis of uncultured patient brain tumor tissue and patient-derived cell lines. The platform has supported engraftment of every patient tumor tested to this point: high- and low-grade adult and pediatric tumor tissue rapidly establishes on OBSCs among endogenous astrocytes and microglia while maintaining the tumor's original DNA profile. Our algorithm calculates dose-response relationships of both tumor kill and OBSC toxicity, generating summarized drug sensitivity scores on the basis of therapeutic window and allowing us to normalize response profiles across a panel of U.S. Food and Drug Administration (FDA)-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment show positive associations to clinical outcomes, suggesting that the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.

Bioengineered Human Pyloric Sphincters Using Autologous Smooth Muscle and Neural Progenitor Cells.

Gastroparesis leads to inadequate emptying of the stomach resulting in severe negative health impacts. Appropriate long-term treatments for these diseases may require pyloric sphincter tissue replacements that possess functional smooth muscle cell (SMC) and neural components. This study aims to bioengineer, for the first time, innervated human pylorus constructs utilizing autologous human pyloric sphincter SMCs and human neural progenitor cells (NPCs). Autologous SMCs and NPCs were cocultured in dual-layered hydrogels and formed concentrically aligned pylorus constructs. Innervated autologous human pylorus constructs were characterized through biochemical and physiologic assays to assess the phenotype and functionality of SMCs and neurons. SMCs within bioengineered human pylorus constructs displayed a tonic contractile phenotype and maintained circumferential alignment. Neural differentiation within bioengineered constructs was verified by positive expression of ßIII-tubulin, neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT). Autologous bioengineered innervated human pylorus constructs generated a robust spontaneous basal tone and contracted in response to potassium chloride (KCl). Contraction in response to exogenous neurotransmitter acetylcholine (ACh), relaxation in response to vasoactive intestinal peptide (VIP), and electrical field stimulation (EFS) were also observed. Neural network integrity was demonstrated by inhibition of EFS-induced relaxation in the presence of a neurotoxin or nNOS inhibitors. Partial inhibition of ACh-induced contraction and VIP-induced relaxation following neurotoxin treatment was observed. These studies provide a proof of concept for bioengineering functional innervated autologous human pyloric sphincter constructs that generate a robust basal tone and contain circumferentially aligned SMCs, which display a tonic contractile phenotype and functional differentiated neurons. These autologous constructs have the potential to be used as (1) functional replacement organs and (2) physiologically relevant models to investigate human pyloric sphincter disorders.

The appendix as a viable source of neural progenitor cells to functionally innervate bioengineered gastrointestinal smooth muscle tissues.

UNLABELLED: Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, but use of these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix would differentiate into enteric neural subsets capable of inducing neurotransmitter-mediated smooth muscle cell (SMC) contraction and relaxation. NPCs were isolated from the appendix and small intestine (SI) of rabbits. Bioengineered internal anal sphincter constructs were developed using the same source of smooth muscle and innervated with NPCs derived from either the appendix or SI. Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry, and functionality was assessed through force-generation studies. Expression of neural and glial differentiation markers was observed in constructs containing appendix- and SI-derived NPCs. The addition of acetylcholine to both appendix and SI constructs caused a robust contraction that was decreased by pretreatment with the neural inhibitor tetrodotoxin (TTX). Electrical field stimulation caused relaxation of constructs that was completely abolished in the presence of TTX and significantly reduced on pretreatment with nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride [l-NAME]). These data indicate that in the presence of identical soluble factors arising from intestinal SMCs, enteric NPCs derived from the appendix and SI differentiate in a similar manner and are capable of responding to physiological stimuli. This coculture paradigm could be used to explore the nature of the soluble factors derived from SMCs and NPCs in generating specific functional innervations. SIGNIFICANCE: This study demonstrates the ability of neural stem cells isolated from the appendix to differentiate into mature functional enteric neurons. The differentiation of neural stem cells from the appendix is similar to differentiation of neural stem cells derived from the gastrointestinal tract. The appendix is a vestigial organ that can be removed with minimal clinical consequence through laparoscopy. Results presented in this paper indicate that the appendix is a potential source of autologous neural stem cells required for cell therapy for the gastrointestinal tract.

Pancreatic Cancer Cells Isolated from Muc1-Null Tumors Favor the Generation of a Mature Less Suppressive MDSC Population.

Mucin 1 (MUC1) is a transmembrane mucin glycoprotein that is over-expressed and aberrantly glycosylated in >80% of human pancreatic ductal adenocarcinoma (PDA) and is associated with poor prognosis. To understand the role of MUC1 in PDA, we have recently developed two mouse models of spontaneous PDA, one that expresses full-length human MUC1 transgene (KCM mice) and one that is null for MUC1 (KCKO mice). We have previously reported that KCM mice express high levels of myeloid derived suppressor cells (MDSCs) in their tumors and develop highly aggressive PDA. To further understand the underlying mechanism for high MDSC levels in KCM-tumors, we generated primary cell lines from KCM and KCKO-tumors. In this study, we report that MDSCs derived using KCM cells express significantly higher levels of arginase 1 and inducible nitric oxide synthase (markers associated with immune suppression) and lower levels of CD115 (a marker associated with maturation of myeloid cells) as compared to KCKO-derived MDSCs. Functionally, KCM-derived MDSCs secrete significantly higher levels of urea and nitric oxide (NO) when co-cultured with normal splenic cells as compared to KCKO-derived MDSCs. Data indicates that KCM-derived MDSCs remain immature and are more suppressive as compared to KCKO-derived MDSCs. This was further corroborated in vivo where MDSCs isolated from KCM-tumor-bearing mice retained their immature state and were highly suppressive as compared to MDSCs derived from KCKO-tumor-bearing mice. Finally, we show that KCM cells secrete significantly higher levels of prostaglandin E2 (PGE2), a COX-2 metabolite and a known driver of suppressive MDSCs as compared to KCKO cells. Thus, inhibiting PGE2 with a specific COX-2 inhibitor reverses the immunosuppressive and immature phenotype of KCM-derived MDSCs. This is the first report that clearly suggests a functional role of pancreatic tumor-associated MUC1 in the development of functional MDSCs.

Tumor necrosis factor-alpha-converting enzyme activities and tumor-associated macrophages in breast cancer.

The role of the tumor microenvironment especially of tumor-associated macrophages (TAMs) in the progression and metastatic spread of breast cancer is well established. TAMs have primarily a M2 (wound-healing) phenotype with minimal cytotoxic activities. The mechanisms by which tumor cells influence TAMs to display a pro-tumor phenotype are still debated although the key roles of immunomodulatory cytokines released by tumor cells, including colony-stimulating factor 1, tumor necrosis factor (TNF) and soluble TNF receptors 1/2, soluble vascular cell adhesion molecule 1, soluble interleukin 6 receptor and amphiregulin, have been demonstrated. Importantly, these factors are released through ectodomain shedding by the activities of the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17). The role of TACE activation leading to autocrine effects on tumor progression has been extensively studied. In contrast, limited information is available on the role of tumor cell TACE activities on TAMs in breast cancer. TACE inhibitors, currently in clinical trials, will certainly affect TAMs and subsequently treatment outcomes based on the substrates it releases. Furthermore, whether targeting a subset of the molecules shed by TACE, specifically those leading to TAMs with altered functions and phenotype, holds greater therapeutic promises than past clinical trials of TACE antagonists' remains to be determined. Here, the potential roles of TACE ectodomain shedding in the breast tumor microenvironment are reviewed with a focus on the release of tumor-derived immunomodulatory factors shed by TACE that directs TAM phenotypes and functions.

Breast tumor cell TACE-shed MCSF promotes pro-angiogenic macrophages through NF-κB signaling.

Most deaths associated with breast cancer, the most common malignancy in women, are caused by metastasis. Tumor associated macrophages significantly contribute to breast cancer progression and development of metastasis through the promotion of angiogenesis which involves a central regulator of macrophage functions: nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Macrophages are activated by macrophage colony stimulating factor (MCSF) and chemokine (C-C motif) ligand 2 (CCL2) to secrete angiogenic factors including vascular endothelial growth factor (VEGF). The release of MCSF from tumor cells is mediated by ectodomain shedding through tumor necrosis factor alpha converting enzyme activation (TACE). Here we determined whether tumor cells TACE-shed MCSF promotes angiogenesis through activation of the NF-κB pathway in macrophages and the subsequent release of VEGF. These interactions were modeled in vitro using a panel of mammary cells mimicking the breast cancer progression from normal murine mammary gland cells to metastatic 4T1 cells along with J774 macrophages, all derived from BALB/c mice. TACE and MCSF expressions were higher in metastatic cells compared to epithelial cells (p < 0.05). Tumor conditioned medias activated the expression of VEGF by macrophages through stimulation of the NF-κB pathway and resulting macrophage secretions that promoted high levels of endothelial cell tubes. Furthermore, the combinations of CCL2, also highly expressed by tumor cells, and MCSF promoted pro-angiogenic macrophages. These results highlight the key role of tumor cell TACE-shed MCSF and secreted CCL2 in stimulating pro-angiogenic macrophages.

Soluble tumor necrosis factor receptors shed by breast tumor cells inhibit macrophage chemotaxis.

Breast tumor cells alter their microenvironment in part through the expression of protumor molecules that influence macrophages during tumor progression and metastasis. Macrophage recruitment is stimulated by chemotactic factors, including tumor necrosis factor alpha (TNF-α), which also stimulates the cytotoxic/tumor cell killing macrophage phenotype. Through TNF-α converting enzyme (TACE/ADAM17) activities, breast tumor cells shed membrane-bound proteins, including their TNF receptors (sTNFR1/2), which serve as decoys sequestering TNF-α and preventing TNF-α-driven apoptosis of tumor cells, thereby decreasing TNF-α bioavailability. Here we investigated the levels of sTNFRs shed by breast tumor cells and determined the effects of shed sTNFRs on macrophage migration toward TNF-α. TNF-α and sTNFRs concentrations were measured in murine normal epithelial, stromal, and mammary tumor cells. The migration of murine macrophages towards TNF-α in the presence of tumor derived soluble factors (TDSFs) shed by TACE was determined. TNF-α concentrations secreted by tumor and normal epithelial cells were below the detection limit contrasting with stromal cells, especially macrophages, which expressed higher levels of TNF-α (P<0.001). Regardless of the cell tested, treatment with the TACE inhibitor TAPI-0 led to a significant decrease in sTNFR2 shed (P<0.05). The dose-dependent macrophage migration toward TNF-α prevented by incubation with TDSFs was not observed with TDSFs collected following TAPI-0 treatment (P<0.05). Furthermore, the TNF-α-driven increased pAkt expression in macrophage was inhibited by TACE shed TDSFs (P<0.05). These results highlight the role of tumor-shed sTNFRs in TNF-α -driven macrophage chemotaxis.

Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases.

Stromal chemokine gradients within the breast tissue microenvironment play a critical role in breast cancer cell invasion, a prerequisite to metastasis. To elucidate which chemokines and mechanisms are involved in mammary cell migration we determined whether mesenchymal D1 stem cells secreted specific chemokines that differentially promoted the invasion of mammary tumor cells in vitro. Results indicate that mesenchymal D1 cells produced concentrations of CCL5 and CCL9 4- to 5-fold higher than the concentrations secreted by 4T1 tumor cells (P < 0.01). Moreover, 4T1 tumor cell invasion toward D1 mesenchymal stem cell conditioned media (D1CM), CCL5 alone, CCL9 alone or a combination CCL5 and CCL9 was observed. The invasion of 4T1 cells toward D1 mesenchymal stem CM was dose-dependently suppressed by pre-incubation with the CCR1/CCR5 antagonist met-CCL5 (P < 0.01). Furthermore, the invasion of 4T1 cells toward these chemokines was prevented by incubation with the broad-spectrum MMP inhibitor GM6001. Additionally, the addition of specific MMP9/MMP13 and MMP14 inhibitors prevented the MMP activities of supernatants collected from 4T1 cells incubated with D1CM, CCL5 or CCL9. Taken together these data highlight the role of CCL5 and CCL9 produced by mesenchymal stem cells in mammary tumor cell invasion.