Mesenchymal Stem Cells Promote Mammosphere Formation and Decrease E-Cadherin in Normal and Malignant Breast Cells

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as "mammospheres" in three-dimensional cultures.We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy

Human Omental-Derived Adipose Stem Cells Increase Ovarian Cancer Proliferation, Migration, and Chemoresistance

Objectives: Adipose tissue contains a population of multipotent adipose stem cells (ASCs) that form tumor stroma and can promote tumor progression. Given the high rate of ovarian cancer metastasis to the omental adipose, we hypothesized that omental-derived ASC may contribute to ovarian cancer growth and dissemination. Materials and Methods: We isolated ASCs from the omentum of three patients with ovarian cancer, with (O-ASC4, O-ASC5) and without (O-ASC1) omental metastasis. BM-MSCs, SQ-ASCs, O-ASCs were characterized with gene expression arrays and metabolic analysis. Stromal cells effects on ovarian cancer cells proliferation, chemoresistance and radiation resistance was evaluated using co-culture assays with luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASCs and control cell lines. SKOV3 cells were intraperitionally injected with or without O-ASC1 to track in-vivo engraftment. Results: O-ASCs significantly promoted in vitro proliferation, migration chemotherapy and radiation response of ovarian cancer cell lines. O-ASC4 had more marked effects on migration and chemotherapy response on OVCA 429 and OVCA 433 cells than O-ASC1. Analysis of microarray data revealed that O-ASC4 and O-ASC5 have similar gene expression profiles, in contrast to O-ASC1, which was more similar to BM-MSCs and subcutaneous ASCs in hierarchical clustering. Human O-ASCs were detected in the stroma of human ovarian cancer murine xenografts but not uninvolved ovaries. Conclusions: ASCs derived from the human omentum can promote ovarian cancer proliferation, migration, chemoresistance and radiation resistance in-vitro. Furthermore, clinical O-ASCs isolates demonstrate heterogenous effects on ovarian cancer in-vitro

Circulating neutrophils and tumor-associated myeloid cells function as a powerful biomarker for response to chemoradiation in locally advanced cervical cancer

Purpose: The immune system’s role in mediating the cytotoxic effects of chemoradiotherapy remains not completely understood. The integration of immunotherapies into treatment will require insight into features and timing of the immune microenvironment associated with treatment response. Here, we investigated the role of circulating neutrophils and tumor-associated myeloid cells (TSAMs) as potential agents and biomarkers for disease-related outcomes in locally advanced cervical cancer (LACC). Material and Methods: Hematologic parameters for two LACC patient cohorts, a retrospective clinical and a prospective translational cohort, were obtained at baseline, weekly during chemoradiotherapy for the retrospective cohort, biweekly during chemoradiotherapy for the prospective cohort, and at the first follow-up visit for both cohorts (mean 14.7 weeks, range 8.1–25.1 weeks for the prospective cohort and 5.3 weeks with a range of 2.7–9.0 weeks for the retrospective cohort). In both cohorts, baseline as well as mean and lowest on-treatment values for platelets, hemoglobin, absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) were analyzed for correlations with disease-related outcomes. In the prospective cohort, circulating myeloid cells were isolated from peripheral blood mononuclear cells (PBMCs), and TSAMs were isolated from tumor tissue via a novel serial cytobrush sampling assay. The samples were analyzed by flow cytometry. Results: In both cohorts, the only hematologic parameter significantly associated with survival was elevated on-treatment mean ANC (mANC), which was associated with lower local failure-free and overall survival rates in the retrospective and prospective cohorts, respectively. mANC was not associated with a difference in distant metastases. CD11b+CD11c- TSAMs, which act as a surrogate marker for intratumoral neutrophils, steadily decreased during the course of chemoRT and nadier’d at week 5 of treatment. Conversely, circulating myeloid cells identified from PBMCs steadily increased through week 5 of treatment. Regression analysis confirmed an inverse relationship between circulating myeloid cells and TSAMs at this time point. Conclusions: These findings identify on-treatment mean neutrophil count as a predictor of disease-related outcomes, suggest that neutrophils contribute to chemoradiation treatment resistance, and demonstrate the importance of techniques to measure intratumoral immune activity

Stromal Cells Derived from Visceral and Obese Adipose Tissue Promote Growth of Ovarian Cancers

<div><p>Obesity, and in particular visceral obesity, has been associated with an increased risk of developing cancers as well as higher rates of mortality following diagnosis. The impact of obesity on adipose-derived stromal cells (ASC), which contribute to the formation of tumor stroma, is unknown. Here we hypothesized that visceral source and diet-induced obesity (DIO) changes the ASC phenotype, contributing to the tumor promoting effects of obesity. We found that ASC isolated from subcutaneous (SC-ASC) and visceral (V-ASC) white adipose tissue(WAT) of lean(Le) and obese(Ob) mice exhibited similar mesenchymal cell surface markers expression, and had comparable effects on ovarian cancer cell proliferation and migration. Obese and visceral derived ASC proliferated slower and exhibited impaired differentiation into adipocytes and osteocytes <i>in vitro</i> as compared to ASC derived from subcutaneous WAT of lean mice. Intraperitoneal co-injection of ovarian cancer cells with obese or visceral derived ASC, but not lean SC-ASC, increased growth of intraperitoneal ID8 tumors as compared to controls. Obese and V-ASC increased stromal infiltration of inflammatory cells, including CD3+ T cells and F4/80+ macrophages. Obese and visceral derived ASC, but not lean SC-ASC, increased expression of chemotactic factors IL-6, MIP-2, and MCP-1 when cultured with tumor cells. Overall, these results demonstrate that obese and V-ASC have a unique phenotype, with more limited proliferation and differentiation capacity but enhanced expression of chemotactic factors in response to malignant cells which support infiltration of inflammatory cells and support tumor growth and dissemination.</p></div

Gating strategy for the detection of different immune cell subsets from malignant ascites of ovarian cancer patients.

Cells collected from ascites were stained with a 9-color flow cytometry panel and analyzed in an X-20 Fortessa flow cytometer. (A) The gating strategy is depicted starting with the detection of lymphocytes, followed by live cells and CD3+ T cells that are separated according to CD4 and CD8 expression. (B) Fluorescence-minus-one (FMO) staining is shown for the different markers analyzed on CD4+ and CD8+ T cells. (PDF)</p

Ascitic fluid cell CDR3β motifs associated with prognosis.

(A) Examples of CD4+ T cell receptor (TCR) motifs associated with different prognoses. (B) Examples of CD8+ TCR motifs associated with different prognoses. The figures show the logo and phylogenetic tree of the clustered CDR3β peptides for each motif. The phylogenetic tree also includes peptides found as the best hit from McPAS; the complete set of motifs associated with excellent or poor/worst prognosis is shown in S7A Table. (C) Association networks constructed to form the cluster of motifs associated with response to platinum therapy. Nodes in the networks indicate individual patients; the color of the node indicates prognosis. Edges in the networks indicate an association between a pair of samples if they share GLIPH specificity groups associated with prognosis. The networks validated the statistical algorithm used to identify prognosis-associated specificity groups.</p

Difference in T cell receptor (TCR) characteristics between ascites of patients grouped in Cluster A and ascites of patients grouped in the remaining clusters (others).

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Increased T cell receptor productive rearrangements in ascites of patients with <i>BRCA1/2</i> tumor mutations.

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Secretion of VEGF, IL-6, MIP2 and MCP1 by ASC cells in ID8 spheroid-CM.

<p>ASC were plated at a density of 20,000/ml in cultured in spheroid medium(control) or medium contained 50% ID8 spheroid-CM for 5 days. Supernatant were collected for Luminex assay. Concentration of VEGF (<b>A</b>) IL-6 (<b>B</b>), MIP2(<b>C</b>) and MCP1 (<b>D</b>).(Shown are mean ± SEM. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01, ***, P<0.001 and ****, P<0.0001. Student <i>t</i> test, two tailed.)</p

ID8 tumor growth pairwise comparison between groups

<p>Shown as Statistic results of ID8 tumor growth between groups based on a linear mixed effect model with repeat measures to evaluate the effect of ASC source (V/SC) and obesity status (obese/lean) versus control group across multiple time points. Note that the difference was between two groups averaged over all time points because there was not significant interaction between treatment group and time point. SE: standard error.</p><p>ID8 tumor growth pairwise comparison between groups</p