POLE-mutated Endometrial "Carcinosarcoma".

The molecular subtype classification of endometrial carcinomas has conceptually changed our approach to this disease. However, open questions remain about how to integrate certain histotype diagnoses with the molecular subtype. We report 2 cases with morphologic suspicion for endometrial carcinosarcoma, that still fell short of the essential criteria for diagnosing carcinosarcoma. On subsequent molecular testing pathogenic POLE mutations were detected and a descriptive diagnosis of endometrial endometrioid carcinomas, low-grade with a homologous sarcoma component was rendered. This challenges the existence of POLE-mutated "carcinosarcoma."

Glioma-derived IL-33 orchestrates an inflammatory brain tumor microenvironment that accelerates glioma progression.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.

Radiotherapy-Activated Cancer-Associated Fibroblasts Promote Tumor Progression through Paracrine IGF1R Activation.

Preoperative radiotherapy (RT) is a mainstay in the management of rectal cancer, a tumor characterized by desmoplastic stroma containing cancer-associated fibroblasts (CAF). Although CAFs are abundantly present, the effects of RT to CAF and its impact on cancer cells are unknown. We evaluated the damage responses of CAF to RT and investigated changes in colorectal cancer cell growth, transcriptome, metabolome, and kinome in response to paracrine signals emerging from irradiated CAF. RT to CAF induced DNA damage, p53 activation, cell-cycle arrest, and secretion of paracrine mediators, including insulin-like growth factor-1 (IGF1). Subsequently, RT-activated CAFs promoted survival of colorectal cancer cells, as well as a metabolic switch favoring glutamine consumption through IGF1 receptor (IGF1R) activation. RT followed by IGF1R neutralization in orthotopic colorectal cancer models reduced the number of mice with organ metastases. Activation of the downstream IGF1R mediator mTOR was significantly higher in matched (intrapatient) samples and in unmatched (interpatient) samples from rectal cancer patients after neoadjuvant chemoradiotherapy. Taken together, our data support the notion that paracrine IGF1/IGF1R signaling initiated by RT-activated CAF worsens colorectal cancer progression, establishing a preclinical rationale to target this activation loop to further improve clinical responses and patient survival.Significance: These findings reveal that paracrine IGF1/IGF1R signaling promotes colorectal cancer progression, establishing a preclinical rationale to target this activation loop. Cancer Res; 78(3); 659-70. ©2017 AACR.

Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident Cardiac Atrial appendage Stem Cells.

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors, but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay, we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts, perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells, specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs), including a clonogenic potential of ~21.5% and expression of pluripotency associated genes like Oct-4, Nanog, c-Myc and Klf-4. Similar to CASCs, migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate, a PDGF receptor inhibitor, suggested a role for the PDGF-AA/PDGF receptor α axis in enhancing the migration process of CASCs. In conclusion, our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors, like PDGF-AA, can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury.

Single cell and spheroid collagen type I invasion assay.

Tumor invasion is the outcome of a complex interplay between cancer cells and the stromal environment and requires the infiltration of a dense, cross-linked meshwork of collagen type I extracellular matrix. We use a membrane-free single-cell and spheroid-based complementary model to study cancer invasion through native collagen type I matrices. Cell morphology is preserved during the assays allowing real-time monitoring of invasion-induced changes in cell structure and F-actin organization. Combination of these models with computerized quantification permits the calculation of highly reproducible and operator-independent data. These assays are versatile in the use of fluorescent probes and have a flexible kinetic endpoint. Once the optimal experimental conditions are empirically determined, the collagen type I invasion assays can be used for preclinical validation of small-molecule inhibitors targeting invasion. Initiation and monitoring of the single-cell and spheroid invasion model can be achieved in 8 h (over 3 days) and in 14 h (over 5 days), respectively.

Bone marrow-derived mesenchymal stem cells promote colorectal cancer progression through paracrine neuregulin 1/HER3 signalling.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSC) migrate to primary tumours and drive tumour progression. This study aimed to identify the molecular mechanisms associated with these heterotypic cellular interactions and analyse their relevance in colorectal cancer (CRC). DESIGN: Paracrine interactions of BM-MSC with CRC cells were studied using collagen invasion assays, cell counts, flow cytometric cell-cycle analysis and tumour xenograft models. The role of neuregulin 1 (NRG1) and the human epidermal growth factor receptor (HER) family pathways were investigated using tyrosine kinase assays, mass spectrometry, pharmacological inhibition, antibody-mediated neutralisation and RNA interference. Transmembrane neuregulin 1 (tNRG1), HER2 and HER3 expression was analysed in primary CRC (n=54), adjacent normal colorectal tissues (n=4), liver metastases (n=3) and adjacent normal liver tissues (n=3) by immunohistochemistry. RESULTS: BM-MSC stimulate invasion, survival and tumorigenesis of CRC through the release of soluble NRG1, activating the HER2/HER3-dependent PI3K/AKT signalling cascade in CRC cells. Similarly, tumour-associated mesenchymal cells (T-MC) in CRC demonstrate high tNRG1 expression, which is significantly associated with advanced Union for International Cancer Control stage (p=0.005) and invasion depth (p=0.04) and decreased 5-year progression-free survival (p=0.01). HER2 and HER3 show membrane localisation in cancer cells of CRC tissue. CONCLUSION: Paracrine NRG1/HER3 signals initiated by BM-MSC and T-MC promote CRC cell progression, and high tNRG1 expression is associated with poor prognosis in CRC.

Differential secretome analysis of cancer-associated fibroblasts and bone marrow-derived precursors to identify microenvironmental regulators of colon cancer progression.

The identification of cancer-associated fibroblast (CAF)-derived proteins that mediate interactions between the tumor stroma and cancer cells is a crucial step toward the discovery of new molecular targets for therapy or molecular signatures that improve tumor classification and predict clinical outcome. CAF are α-smooth muscle actin positive, representing a myofibroblast phenotype that may differentiate from multiple precursor cells, including bone marrow-derived mesenchymal stem cells (MSC). Transforming growth factor-ß1 (TGF-ß1) is a crucial inducer of α-smooth muscle actin positive CAFs. In this study, we aimed to identify CAF-derived regulators of colon cancer progression by performing a high-throughput differential secretome profiling between CAF compared to noncancer-activated bone marrow-derived MSC. In addition, we explored the effect of TGF-ß1 on the secretion of proteins by bone marrow-derived MSC in comparison with the protein secretion profile of CAF. TGF-ß1 induced de novo secretion of 84 proteins in MSC, of which 16 proteins, including stromal-derived factor-1α and Rantes, were also present in CAF secretome. Immunohistochemistry further validated the expression of selected candidates such as tenascin C, fibronectin ED-A domain and stromal-derived factor-1 in clinical colon cancer specimens. In conclusion, this differential secretome approach enabled us to identify a series of candidate biomarkers for colon cancer that are associated with a CAF-specific phenotype.

Resident and bone marrow-derived mesenchymal stem cells in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major healthcare problem worldwide affecting more than half a million patients each year. Despite considerable advances in the treatment of HNSCC, a high rate of recurrences aggravates the clinical situation and disease outcomes have only modestly improved. Recent insights show that cancer is not only a disease of the transformed epithelium but is also influenced and dependent on its stromal environment. In this review we suggest that resident and bone marrow (BM)-derived mesenchymal stem cells (MSCs) are precursors of the stroma associated with HNSCC and contribute to blood- and lymph angiogenesis, modulate the immune system and produce tumor-associated myofibroblasts. In addition, the impact of radiation therapy on the stromal reaction in HNSCC is discussed. Understanding the mechanisms of how MSCs promote invasive growth and metastasis in HNSCC and respond to cancer management strategies is of profound medical importance and will help us to design improved therapeutic protocols.

Modeling and quantification of cancer cell invasion through collagen type I matrices.

Tumor invasion is the outcome of a complex interplay between cancer cells and the stromal environment. Considering the contribution of the stromal environment, we developed a membrane-free single-cell and spheroid based complementary model to study cancer invasion through native collagen type-I matrices. Cell morphology is preserved during the assays allowing real time monitoring of invasion-induced changes in cell structure and F-actin organization. Combining these models with computerized quantification permits the calculation of highly reproducible and operator-independent data. These assays are versatile in the use of fluorescent probes and have a flexible kinetic endpoint. Once the optimal experimental conditions are empirically determined, the collagen type-I invasion assays can be used for preclinical validation of small-molecule inhibitors targeting invasion. Initiation and monitoring of the single-cell and spheroid invasion model can be achieved in 8 h (over 3 days) and in 14 h (over 8 days) respectively.

The extracellular matrix regulates cancer progression and therapy response: implications for prognosis and treatment.

Emerging evidence points towards a key role of the extracellular matrix (ECM) during tumor progression and therapy resistance. Paradoxically, in today's routine of cancer management the ECM is not taken into account. It is the aim of the present review to broaden our understanding of the mechanisms of therapy resistance, taking the ECM as a presumptive central regulator. The stromal ecosystem drives the accumulation of ECM at the invasion front. Therefore, we address the question whether the detection of ECM signatures in histopathology and biofluids may help predicting therapy resistance and determining the prognosis of cancer. Since the ECM is an attractive target for tumor therapy, current therapeutic strategies in preclinical or clinical development will be discussed.