Triple negative breast cancer (TNBC) is a clinical therapeutic challenge due to the lack of receptors for estrogen, progesterone, and human epidermal growth factor receptor 2 which limits treatment options to chemotherapy and radiation. With current standard therapy less than 30% will survive the 5-year remission rate, especially Hispanic and African-American women where TNBC is more frequent and has the lowest (<14%) 5-year survival rates. Recent studies indicate that Hedgehog (Hh) signaling is active and correlates with reduced survival rates in TNBC patients. However, the role of the Hh signaling in the breast tumor microenvironment is not well understood. A main cellular mediator of Hh signaling is the adjacent mesenchyme which promotes tumor growth via a paracrine interaction, but which is also highly heterogeneous. As mesenchymal sub-types have been associated to specific pharmacological therapies, understanding of the mechanisms in mesenchymal-driven tumors is necessary for combinatorial pharmacological treatments that can increase survival rates and eliminate tumor relapse in patients. We developed a tumor-mesenchymal in vitro model to evaluate the role of mesenchymal cell sub-types from different sources in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. Cells were culture in adjacent compartments and active Hh signaling was confirmed by up-regulation of canonical Hh target genes Gli1, Patch1 and SMO. As a source of mesenchymal cell sub-types we evaluated TGF-β treated fibroblasts, cancer- associated and normal breast primary fibroblasts, and tumor epithelial cells that undergo epithelial-mesenchymal transition (EMT). Paracrine Hh signaling significantly increased proliferation (5-10%) of tumor cells after 72-96hrs in co-culture. Cells that undergo EMT promoted cell growth at higher rates than myofibroblasts. SHH treatment alone (without TGF-β pre-treatment) had an inhibitory effect in the proliferation rates of the adjacent epithelium compartment. Observed increase in tumor cell proliferation was abolished when treated with the Hh signaling inhibitor cyclopamine. Stem cell surface markers CD44+CD24- remained unchanged but a significant increase was observed in the proliferating CD44+/CD24+ sub-population. Similar results were observed in normal human breast cells suggesting that activation of Hh signaling in the mesenchyme is sufficient to promote cell growth in the epithelium. Computational analysis of a panel of genes associated to Hh signaling in breast cancer patients (>500 samples) show a strong correlation among TNBC patients and mesenchymal-driven Hh signaling in >80% of samples. We evaluated the distribution of mesenchymal cell-sub-types and stem cells among the sub-set of basal TNBC samples (82 samples). Gene markers associated to myofibroblasts, EMT, mesenchymal stem cell and cancer stem cell were found in samples with active Hh signaling and co-expressed in groups of two or more which represented the 45.6% of the total samples. The most abundant cell sub-type were cancer stem cells with 43% of the total samples and alone by itself in 13.9% of samples. Together, these results indicate that the Hh-pathway and adjacent tumor microenvironment are key players in the progression of TNBC. Mesenchymal cell component can modulate tumor growth behavior induced by Hh signaling and is a potential new clinical target for TNBC. Citation Format: Maribella Domenech, Karla P. Ramos, Wandaliz Torres, Fernando Boria. Mesenchymal-driven hedgehog signaling drives tumor cell growth and is a potential new target for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B05.

Methods: Ultrasound contrast microbubbles were prepared from decafluorobutane gas and stabilized with a lipid monolayer shell, decorated with PEG brush and biotin. sLe a -polyacrylamide-biotin (or a control carbohydrate-free polymer) was attached to microbubbles via a streptavidin linker. Activated human platelets were attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets, platelet dishes blocked by an anti-p-selectin antibody, and dishes coated by recombinant p-selectin were also prepared. Microbubble adhesion to these target surfaces was assessed in an inverted parallel plate flow chamber, with wall shear stress at 40, 30, 20, 15, 10 and 5 dynes cm À2 . Results: Targeted microbubbles were binding and then rolling on the surface of platelets and p-selectin-coated dishes. The capture efficiency (AESEM) of sLe a -bubbles on platelet-coated dishes (n¼8) was 15.73AE0.40% at 5 dynes cm À2 , 10.57AE0.86% at 10 dynes cm À2 , 6.66AE0.47% at 20 dynes cm À2 , 4.79AE0.24% at 30 dynes cm À2 and 3.56AE0.25% at 40 dynes cm À2 . The capture efficiency of sLe a -bubbles on p-selectin-coated dishes (n¼4) was comparable: 21.84AE1.65% at 5 dynes cm À2 , 11.16AE1.28% at 10 dynes cm À2 , 5.89AE0.95% at 20 dynes cm À2 , 3.87AE0.12% at 30 dynes cm À2 and 3.42AE0.10% at 40 dynes cm À2 . No adhesion of control sLe a -free bubbles (n¼3) was observed. There was binding neither on fibrinogen-coated dishes without platelets (n¼3) nor on platelet dishes blocked by an anti-pselectin antibody (n¼2). Conclusion: Microbubble targeting using fast binding ligands like the selectin-ligand sLe a is a promising and effective strategy for the construction of ultrasound contrast agents targeted to activated platelets. In our study, we demonstrate specific binding of microbubbles to p-selectin at high shear flow conditions. Thus, non-invasive imaging of activated platelets on atherosclerotic plaques can be feasible.

Recent studies correlate Hedgehog (Hh) signaling with reduced survival rates in triple negative breast cancer (TNBC) patients. Activation of hedgehog signaling in the adjacent mesenchyme has been shown to promote tumor growth and it is a poor prognosis factor for TNBC. We developed a novel tumor-mesenchymal in vitro model of hedgehog signaling in TNBC to evaluate the role of mesenchymal cell sub-populations in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. As a source of mesenchymal cell sub-populations we evaluated myofibroblasts, mesenchymal stem cells derived from bone marrow or adipose tissue and tumor cells that undergo epithelial-mesenchymal transition (EMT). Tumor cells were culture with 1 or 2 mesenchymal cell sub-populations in adjacent compartments +/- SHH ligand for 72-96hrs. Active Hh signaling was confirmed by up-regulation of main Hh target genes (e.g. Gli1, Patch1). Paracrine Hh signaling only in bone marrow-derived mesenchymal stem cell and EMT significantly increased proliferation (10-15%) of TNBC and normal breast cancer cell lines. Addition of adipose-derived mesenchymal stem cells or pharmacological Hh inhibitors (Cyclopamine and Gant61) partially abolished tumor growth indicating that other non-canonical signals are involve in paracrine Hh-driven tumors. Cancer stem cell markers were selectively modulated in co-cultures. Our results suggest that paracrine Hh signaling-driven by bone marrow and EMT mesenchymal are potential therapeutic targets to treating TNBC. Citation Format: Karla P. Ramos, Maribella Domenech. Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1334. doi:10.1158/1538-7445.AM2017-1334

The development and progression of tumors are modulated by the interaction among multiple cell types present at the tumor adjacent tissue. Few in vitro cell culture platforms allow one to test multi-cellular interactions and those suitable require high numbers of cells limiting studies to cell lines and excluding heterogeneous patient samples, and fail to provide tunable discrete adjacent compartments for the retrieval of independent cell population readouts. Mixing of cells in adjacent compartments and cell sorting after mixed culture represent a costly and time consuming procedure that often results is lost, reduction and/or damage of the recovered sample. To overcome some of these limitations we have developed an open multi-microwell array that enables co-culture of up to 4 cell populations in discrete adjacent wells using either 2- or 3-dimension culture conditions. The circular microwells are made of poly-dimethyl siloxane (PDMS) and placed on top of glass or tissue culture plastic. The open-well format allows cells and culture media to be manually loaded and replaced using a pipette independently of passive pumping method, and without the need of vacuum-assisted filling or syringe pumps. The microwell array is aligned to a 96well plate format for automated fluorescent readouts. The cell culture surface area of each microwell is 5.7mm2 (5ul) and can accommodate 5,000-10,000 cells but the design of each microwell can be scale-down to accommodate smaller cell samples. The adjacent microwells are contained within an outer-ring of 8mm diameter and 750um height. After cell seeding, adjacent compartments can be connected by dispensing a volume of 30-40ul inside the outer ring. A model of tumor-mesenchyme paracrine Hedgehog signaling was developed and characterized to show the feasibility of this array for multi-cell soluble factor signaling studies. Activation of Hedgehog (Hh) signaling in the mesenchyme promotes proliferation of tumor cells via secreted factors. As a source of mesenchymal cell sub-types we evaluated TGF-b treated fibroblasts and tumor epithelial cells that undergo epithelial-mesenchymal transition. Myofibroblast phenotype was confirmed based on overexpression of smooth muscle actin, vimentin and fibroblast activating protein, and reduce levels of caveolin-1. Active Hh signaling and pharmacological inhibition with cyclopamine were confirmed by up- and down- regulation of canonical Hh target genes Gli1 and Patch1. Active hedgehog signaling and cell viability was maintained over 5-7days 2-dimensional and collagen type I embedded mesenchymal cells. Breast-derived human epithelial and mesenchymal cells co-cultured in microwells and transwells showed similar increase in tumor cell proliferation rates but with a reduction of 80% or more in the total cells needed compared to transwells. Flow cytometer read-outs of proliferation and tumor cell distribution of CD44/CD24 cell surface receptors were similar among transwells and microwells. Adjacent culture of mouse derived fibroblasts (NIH-3T3) and human tumor cells showed recovery of genomic sample without cell-population mixing using primers for specific for human and mouse GAPDH. Doubling of the number of microwells with mesenchymal cells modulated the observed proliferative effect in tumor cells suggesting that mesenchymal cell ratios modulate tumor response to paracrine Hh signaling. This observation could not be replicated in macro-scale platforms as active Hh signaling in the mesenchyme is achieved via cell growth arrest using a combination of low serum and cell confluence. The open multi-microwell array represents a new user-friendly multi-culture microscale array with optimized capabilities for the study of paracrine signaling among discrete cell populations compared to macroscale culture technology. Citation Format: Karla P. Ramos, Fernando Boria, Maribella Domenech. Open multi-microwell array for the study of paracrine signaling in tumors. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B02.