Travis Solley

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

Introduction 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. Objective 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. Results 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. Conclusions 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.

Omental adipose tissue-derived stromal cells promote vascularization and growth of endometrial tumors.

Purpose: Adipose tissue contains a population of tumor-tropic mesenchymal progenitors, termed adipose stromal cells (ASC), which engraft in neighboring tumors to form supportive tumor stroma. We hypothesized that intra-abdominal visceral adipose tissue may contain a uniquely tumor-promoting population of ASC to account for the relationship between excess visceral adipose tissue and mortality of intra-abdominal cancers. Experimental Design: To investigate this, we isolated and characterized ASC from intra-abdominal omental adipose tissue (O-ASC) and characterized their effects on endometrial cancer progression as compared with subcutaneous adipose-derived mesenchymal stromal cells (SC-ASC), bone marrow–derived mesenchymal stromal cells (BM-MSC), and lung fibroblasts. To model chronic recruitment of ASC by tumors, cells were injected metronomically into mice bearing Hec1a xenografts. Results: O-ASC expressed cell surface markers characteristic of BM-MSC and differentiated into mesenchymal lineages. Coculture with O-ASC increased endometrial cancer cell proliferation in vitro. Tumor tropism of O-ASC and SC-ASC for human Hec1a endometrial tumor xenografts was comparable, but O-ASC more potently promoted tumor growth. Compared with tumors in SC-ASC–injected mice, tumors in O-ASC–injected mice contained higher numbers of large tortuous desmin-positive blood vessels, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. O-ASC exhibited enhanced motility as compared with SC-ASC in response to Hec1a-secreted factors. Conclusions: Visceral adipose tissue contains a population of multipotent MSCs that promote endometrial tumor growth more potently than MSCs from subcutaneous adipose tissue. We propose that O-ASCs recruited to tumors express specific factors that enhance tumor vascularization, promoting survival and proliferation of tumor cells. Clin Cancer Res; 18(3); 771–82. ©2011 AACR.

Histone deacetylase inhibitors stimulate dedifferentiation of human breast cancer cells through WNT/β-catenin signaling

Recent studies have shown that differentiated cancer cells can dedifferentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti‐cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self‐renewal and expansion of hematopoietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells toward the more resistant stem cell‐like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA‐231, were sorted based on aldehyde dehydrogenase (ALDH) activity and subsequently ALDH‐negative and ALDH‐positive cells were treated with one of two known HDAC inhibitors, VA or suberoylanilide hydroxamic acid. In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single‐cell‐sorted ALDH‐negative cells spontaneously generated ALDH‐positive cells in vitro. Treatment of ALDH‐negative cells with HDAC inhibitors promoted the expansion of ALDH‐positive cells and increased mammosphere‐forming efficiency. Most importantly, it significantly increased the tumor‐initiating capacity of ALDH‐negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β‐catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC‐inhibitor‐induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer. STEM CELLS2012;30:2366–2377

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.

The Antihelmintic Drug Pyrvinium Pamoate Targets Aggressive Breast Cancer

WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.

Mesenchymal stem cells mediate the clinical phenotype of inflammatory breast cancer in a preclinical model

IntroductionInflammatory breast cancer (IBC) is an aggressive type of breast cancer, characterized by very rapid progression, enlargement of the breast, skin edema causing an orange peel appearance (peau d’orange), erythema, thickening, and dermal lymphatic invasion. It is characterized by E-cadherin overexpression in the primary and metastatic disease, but to date no robust molecular features that specifically identify IBC have been reported. Further, models that recapitulate all of these clinical findings are limited and as a result no studies have demonstrated modulation of these clinical features as opposed to simply tumor cell growth.MethodsHypothesizing the clinical presentation of IBC may be mediated in part by the microenvironment, we examined the effect of co-injection of IBC xenografts with mesenchymal stem/stromal cells (MSCs).ResultsMSCs co-injection significantly increased the clinical features of skin invasion and metastasis in the SUM149 xenograft model. Primary tumors co-injected with MSCs expressed higher phospho-epidermal growth factor receptor (p-EGFR) and promoted metastasis development after tumor resection, effects that were abrogated by treatment with the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. E-cadherin expression was maintained in primary tumor xenografts with MSCs co-injection compared to control and erlotinib treatment dramatically decreased this expression in control and MSCs co-injected tumors. Tumor samples from patients demonstrate correlation between stromal and tumor p-EGFR staining only in IBC tumors.ConclusionsOur findings demonstrate that the IBC clinical phenotype is promoted by signaling from the microenvironment perhaps in addition to tumor cell drivers.

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

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 in vitro 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.

Abstract 177: Obesity promotes growth of ovarian cancer through adipose stem cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Intraperitoneal spread to visceral adipose is a frequent event and a major cause of morbidity and mortality in women with ovarian cancer. Excess visceral adiposity increases the risk of ovarian cancer. We hypothesized that obesity effects may be mediated by the adipose-derived stem cell population (ASC) to enhance the initiation and progression of ovarian cancer. Results: Intraperitoneally injected ID8 ovarian cancer cells grew significantly faster in mice with diet-induced obesity (p<0.05). To determine if ASC mediated this effect, ASC were then isolated from the subcutaneous (SC-ASC) and visceral adipose (V-ASC) of mice with diet-induced obesity and lean mice. ASC from all sources expressed same mesenchymal cell surface markers and similarly increased ID8 cell proliferation and survival following paclitaxel treatment. There was no significant difference in ID8 migration in response to ASC conditioned media from lean and obese mice. ASC from obese mice exhibited impaired differentiation into adipocytes and osteocytes in-vitro as compared to ASC from lean mice. 106 renilla luciferase expressing ID8 were co-injected intra-peritoneally with equal number of different ASC isolates into C57Bl6 mice. SC-ASC from obese mice promoted growth of intraperitoneal tumor and the formation of metastasis as compared to SC-ASC from lean mice (p<0.05). There was no significant difference in the effect of V-ASCs from obese and lean mice on ID8 cells. There was trend towards more rapid tumor growth in mice injected with V-ASC as compared to SC-ASC from lean mice. Ascites volume was highest in mice injected with SC-ASC derived from obese mice. SC-ASC from obese mice increased tumor vascularity while ASC from obese mice increased the formation of intra-tumoral perilipin expressing adipocytes. Conclusions: SC-ASC from obese mice promoted tumor growth more than ASC from lean mice suggesting that obesity alters the ASC population to enhance tumor-promoting effects. Obese derived ASC exhibit impaired differentiation potential, which may be linked to tumor promoting effects. Future studies will focus on understanding the mechanisms by which obesity alters the ASC population to impact tumor progression. Citation Format: Yan Zhang, Travis Solley, Karen Lu, Caimiao Wei, Alesandra Nowicka, Ann Klopp. Obesity promotes growth of ovarian cancer through adipose stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 177. doi:10.1158/1538-7445.AM2014-177

Abstract B73: Human omental-derived adipose stem cells: Modulator of ovarian cancer proliferation, migration and chemoresistance

Objectives: Adipose tissue contains a population of multipotent mesenchymal stromal cells (ASC) which exhibit tumor tropism, similar to bone marrow derived mesenchymal stem cells (MSC). Excess visceral adipose tissue increases the risk of ovarian cancer. The omentum is a prominent site for ovarian cancer metastasis. We hypothesize that the omentum serves as a source of ASC which promote ovarian cancer progression. Materials and Methods: ASC were isolated from the omentum (O-ASCs) of three patients with ovarian cancer. O-ASC1 was isolated from a patient with synchronous adenocarcinoma of endometrial and ovarian cancer without peritoneal metastasis; and O-ASC4 and O-ASC5 were isolated from patients with peritoneally disseminated serous ovarian cancer. Gene expression array profiling was performed with using Nimblegen arrays (Roche NimbleGen, Inc., Madison, WI). The impact of stromal cells on proliferation and chemoresistane and radio-protection of ovarian cancer cells was tested with co-culture assays using luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASC and control cell lines. To evaluate of O-ASCs tumor tropism in-vivo experiment was performed. Results: Human O-ASCs were detected engrafted within the stroma of human ovarian cancer xengrafts. O-ASC significantly promoted in-vitro proliferation and migration of ovarian cancer cell lines OVCA 429, OVCA 433, A2780. Co-culture of ovarian cancer cells with O-ASCs increased resistance to chemotherapeutic drugs and radiation. Gene expression array analysis revealed significant differences in expression profiles for group of 415 genes in sub-populations of O-ASCs. Conclusions: Adipose stem cells derived from human omentum promoted the proliferation, migration, chemoresistance and radioresistance of ovarian cancers. Clinical isolates demonstrate heterogenous effects in- vitro. Future studies will determine if tumor promoting effects of O-ASC can be predicted on the basis of clinical or disease related characteristics. Citation Format: Aleksandra Nowicka, Travis Solley, Frank C. Marini, Hadley J. Sharp, Russell R. Broaddus, Kolonin G. Mikhail, Samuel C. Mok, Wendy A. Woodward, Karen H. Lu, Ann H. Klopp. Human omental-derived adipose stem cells: Modulator of ovarian cancer proliferation, migration and chemoresistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B73.

Abstract B69: Mesenchymal stem cells promote tumor-skin involvement in inflammatory breast cancer models

Background: Inflammatory breast cancer (IBC) is an aggressive variant of breast cancer characterized by blocked breast lymphatics and clinically apparent involvement of the skin resulting in erythema, swelling and a high rate of resistance to therapy. Tumor-skin involvement in IBC patients, despite being a rare event, is an event that develops very rapidly, can spread throughout the upper body (chest and back) and ultimately results in breast cancer death. Very little is known about this event and how to prevent it. Mesenchymal stem cells (MSC) are multipotent progenitor cells which are found in normal tissues, including the bone marrow and adipose tissue. MSC exhibit a unique tropism for tumors, where they engraft, form tumor stroma, and alter the tumor microenvironment. MSC have been shown to increase the growth of certain cancers and the incidence of breast xenograft metastasis. Whether MSC directly modify the primary tumor microenvironment or the pre-metastatic niche remote from the primary tumor has not been addressed in IBC tumor models or patients. Methods and Results: Many IBC xenografts grow tumors but do not faithfully recapitulate the skin phenotype of IBC. In order to investigate whether direct exposure to MSC or paracrine exposure using the MSC-conditioned media (MSC-CM) would influence skin involvement and metastasis development and with this recapitulate the IBC phenotype in pre-clinical models, we performed two different in vivo experiments. In the first, we labeled SUM149 cells with luciferase reporter gene and then injected them into the cleared mammary fat pads of female immunocompromised mice (SCID/Beige) with or without 10% MSC (obtained from healthy donors). Transplants were allowed to grow until 300 mm3 in volume and then resected in a survival surgery. At resection time we found that there was a significant difference in the tumor-skin involvement (p=0.0189, Fishers exact test) between the 0% MSC group (4/20) and 10% MSC group (11/18). Furthermore, we assessed metastasis development with live bioluminescence imaging and identified unexpected development of metastasis in the upper body skin plexus (31.6% vs. 58.8%, p = NS). In vitro, MSC co-culture is associated with increased EGFR signaling, and tumors in MSC co-injected animals had increased p-EGFR staining by IHC (70.6% vs. 50.0%, p =0.0455). In a second experiment, we cultured in vitro SUM149 cells (labeled with luciferase reporter gene) with or without MSC-CM for 5 days and then injected them into the cleared mammary fat pads of female immunocompromised mice (SCID/Beige), to determine whether the observed tumor-skin involvement was an effect of MSC secreted factors. Transplants were allowed to grow until 300 mm3 in volume and then resected in a survival surgery. MSC-CM increased significantly the tumor-skin involvement (p=0.0227, Fishers exact test), between the control group (4/16) and MSC-CM group (9/13). We followed up metastasis development with live bioluminescence imaging after tumor resection and observed that several mice developed metastasis in the upper body skin plexus (20.0% vs. 27.2%, p=NS). Conclusion: Our studies show that MSC and MSC-CM increase tumor-skin involvement and metastasis development in an IBC pre-clinical model independent of growth rate. Understanding the role of MSC on the primary tumor microenvironment and the pre-metastatic niche is critical to further develop therapies that target and suppress the tumor-skin involvement and the rapid development of metastasis that IBC patients experience. Citation Format: Lara Lacerda, Daniel Smith, Bisrat Debeb, Travis Solley, Richard Larson, Wei Xu, Savitri Krishnamurthy, Naoto Ueno, Ann Klopp, Wendy Woodward. Mesenchymal stem cells promote tumor-skin involvement in inflammatory breast cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B69.