Search

PDF file - 287K, Figure S1: Photomicrographs of cells in this study. HMEC, HCT116/GFP and MDAmb231 cells were grown to 70-80% confluency. Samples were then viewed with an automated Zeiss Observer Z.1 inverted microscope through a 10x /0.3NA or 20x/0.4NA objective and a phase contrast ring. Images were produced using the AxioCam MRm CCD camera and Axiovision version 4.6 software suite. (Carl Zeiss Inc., Germany).

PDF file - 241K, Figure S7: Segmentation and analysis of vasculature In vivo. In vivo vessels and tumors were independently segmented and quantified within four ordinal quadrants emanating from the tumor centroid as described in Methods.

PDF file - 43K, Figure S5: Relation between tumor growth and pH for all 5 mice. Data for all 5 mice were parsed as in figure 2D wherein the growth (in pixels) and pH were calculated every 22.5o of arc. These data show that growth increased with decreasing pH for all animals, and that there was a threshold of pH above which no growth occurred. Lines represent the trend below threshold for each mouse.

PDF file - 77K, Figure S4: Quantification of tumor growth. Images are shown for mouse tumor #23 on day 4 (green) and day 14 (red). Images were co-registered using the ring clip of the DWC as a fiducial. The centroid of the tumor on day 4 was calculated and radial lines were drawn every 22.5o of arc, beginning at the ring. Growth was calculated as the number of pixels that the day 14 tumor extended beyond the edge of the day 4 tumor using Definiens Developer XD�.

PDF file - 45K, Figure S2: Comparison of normal vs tumor cells by size. Using these phase contrast images, ROI were chosen and area of cells was determined by pixel number using Image-Pro Plus v6.2. Once area was measured, data analysis revealed statistical significance (p

PDF file - 50K, Figure S6: Ex vivo comparison of vessel density in stroma vs tumor tissue. Using the Image Scope v7.1 regions of viable tumor were manually selected. 200�m micrometers were placed around the circumference of the tumor region perpendicular to the tumor edge. A second manually selected region was drawn around the perimeter of the micrometers. This effectively segments the entire tissue section into three regions of interest; the tumor, the edge within 200�m of the tumor and the distant stromal region. The Aperio (Vista, CA, USA) microvessel algorithm v1.0 was used with the following parameters (smoothing 2; dark threshold160; light threshold 210; regioning 6; completion 7) to identify the vascular density which was defined as the vessel area divided by total area (�m2).

PDF file - 88K

PDF file - 45K, Figure S8: In vivo relation between vessel density and tumor growth. The data from Figure S5 were used to compare tumor growth (change in number of pixels from day 4 to day 13) to vessel density (on day 4) in each of the 4 quadrants.

PDF file - 46K, Figure S3: Growth of MDAmb231 tumor within the dorsal window chamber. Mean growth of MDAmb23/GFP tumors within the dorsal window chamber. Cells were embedded in a 0.8 mg/mL collagen type 1 matrix followed by inoculation into the window chamber. Tumor area was measured at days 2, 3, and 16 by counting the number of pixels. Tumor area measured by fluorescence pixel number obtained using Image-Pro Plus 6.2.

Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in acidic extracellular microenvironment. Acidity in the microenvironment is a critical stress factor and selection force for the evolution of aggressive tumor types. There are several acid sensing cell surface receptors and ion channels (ASICs) that can sense acidity in the microenvironment; among them proton sensing G protein-coupled receptors (GPCRs) such as OGR1 (GPR68), TDAG8 (GPR65), GPR4 and GPR132 form a major class of acid receptors. Our previous studies demonstrated that acid adaptation is associated with survival mechanisms like chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. When grown under acidic pH, breast cancer cells accumulate lipids as revealed by staining with Nile Red and perilipin 2, a protein that coats adiposomes or lipid droplets. These are dynamic organelles that store neutral lipids surrounded by a shell of proteins and a phospholipid monolayer. The lipids stored in adiposomes are produced de novo, as acid-induced lipogenic phenotype is maintained, even if cells are grown with de-lipidated serum. Inhibition of fatty acid synthesis was selectively toxic under acidic conditions and attenuated adiposome accumulation. Among the acid sensing receptors, TDAG8 and OGR1 are highly expressed in a panel of breast cancer cell lines compared to non-malignant breast epithelial cells. Highly invasive and acid tolerant MDA-MB-231 cells express significantly higher levels of TDAG8 while MCF7 and T47D cells have high levels of OGR1. We investigated the role of these receptors in transducing the acid signal that results in the accumulation of lipid droplets. CRISPR/Cas9 mediated depletion of the major acid sensors in breast cancer cell lines MCF7 and T47D showed that depletion of OGR1, not TDAG8, specifically inhibited acid induced adiposome accumulation. Additionally, inducing the cells with ogerin, an allosteric activator of OGR1 resulted in adiposomogenesis. Further, OGR1 knock out cells showed inhibition in cell growth under acidic growth conditions compared to OGR1 expressing cells. OGR1 knockout cells were defective in acid induced autophagy. OGR1 is coupled with Gq/11 and, upon ligand (H+) binding, triggers activation of phospholipase C stimulating the formation of the second messenger IP3. Adiposome formation was inhibited in presence of PLC inhibitors, Edelfosine or U73122. These results indicate that OGR1 is the major acid sensing GPCR in these cells that mediate adiposomogenesis. Accumulation of adiposomes is a highly regulated process related to storing autophagic products, and appears to be important in cell survival in acid stress. Taken together, increased dependence on lipid metabolism by cancer cells under acidic conditions reveals novel therapeutic vulnerabilities. Citation Format: Smitha Pillai, Michael Langsen, Jonathan Nguyen, Jonathan Wojtkowiak, Marilyn Bui, Robert Gatenby, Robert Gillies. Acid sensing G protein-coupled receptor OGR1 is required for acid induced adiposomogenesis in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1846.

Certain farnesyl diphosphate (FPP) analogs are potent inhibitors of the potential anticancer drug target protein farnesyltransferase (FTase), but these compounds are not suitable as drug candidates. Thus, phosphoramidate prodrug derivatives of the monophosphate precursors of FPP-based FTase inhibitors have been synthesized. The monophosphates themselves were significantly more potent inhibitors of FTase than the corresponding FPP analogs. The effects of the prodrug 5b (a derivative of 3-allylfarnesyl monophosphate) have been evaluated on prenylation of RhoB and on the cell cycle in a human malignant schwannoma cell line (STS-26T). In combination treatments, 1-3 microM 5b plus 1 microM lovastatin induced a significant inhibition of RhoB prenylation, and a combination of these drugs at 1 microM each also resulted in significant cell cycle arrest in G1. Indeed, combinations as low as 50 nM lovastatin + 1 microM 5c or 250 nM lovastatin + 50 nM 5c were highly cytostatic in STS-26T cell culture.