Prostate fibroblasts and prostate cancer associated fibroblasts exhibit different metabolic, matrix degradation and PD-L1 expression responses to hypoxia.

Fibroblasts are versatile cells that play a major role in wound healing by synthesizing and remodeling the extracellular matrix (ECM). In cancers, fibroblasts play an expanded role in tumor progression and dissemination, immunosuppression, and metabolic support of cancer cells. In prostate cancer (PCa), fibroblasts have been shown to induce growth and increase metastatic potential. To further understand differences in the functions of human PCa associated fibroblasts (PCAFs) compared to normal prostate fibroblasts (PFs), we investigated the metabolic profile and ECM degradation characteristics of PFs and PCAFs using a magnetic resonance imaging and spectroscopy compatible intact cell perfusion assay. To further understand how PFs and PCAFs respond to hypoxic tumor microenvironments that are often observed in PCa, we characterized the effects of hypoxia on PF and PCAF metabolism, invasion and PD-L1 expression. We found that under normoxia, PCAFs displayed decreased ECM degradation compared to PFs. Under hypoxia, ECM degradation by PFs increased, whereas PCAFs exhibited decreased ECM degradation. Under both normoxia and hypoxia, PCAFs and PFs showed significantly different metabolic profiles. PD-L1 expression was intrinsically higher in PCAFs compared to PFs. Under hypoxia, PD-L1 expression increased in PCAFs but not in PFs. Our data suggest that PCAFs may not directly induce ECM degradation to assist in tumor dissemination, but may instead create an immune suppressive tumor microenvironment that further increases under hypoxic conditions. Our data identify the intrinsic metabolic, ECM degradation and PD-L1 expression differences between PCAFs and PFs under normoxia and hypoxia that may provide novel targets in PCa treatment.

Metabolic fingerprinting by nuclear magnetic resonance of hepatocellular carcinoma cells during p53 reactivation-induced senescence.

Cellular senescence is characterized by stable cell cycle arrest. Senescent cells exhibit a senescence-associated secretory phenotype that can promote tumor progression. The aim of our study was to identify specific nuclear magnetic resonance (NMR) spectroscopy-based markers of cancer cell senescence. For metabolic studies, we employed murine liver carcinoma Harvey Rat Sarcoma Virus (H-Ras) cells, in which reactivation of p53 expression induces senescence. Senescent and nonsenescent cell extracts were subjected to high-resolution proton (1H)-NMR spectroscopy-based metabolomics, and dynamic metabolic changes during senescence were analyzed using a magnetic resonance spectroscopy (MRS)-compatible cell perfusion system. Additionally, the ability of intact senescent cells to degrade the extracellular matrix (ECM) was quantified in the cell perfusion system. Analysis of senescent H-Ras cell extracts revealed elevated sn-glycero-3-phosphocholine, myoinositol, taurine, and creatine levels, with decreases in glycine, o-phosphocholine, threonine, and valine. These metabolic findings were accompanied by a greater degradation index of the ECM in senescent H-Ras cells than in control H-Ras cells. MRS studies with the cell perfusion system revealed elevated creatine levels in senescent cells on Day 4, confirming the 1H-NMR results. These senescence-associated changes in metabolism and ECM degradation strongly impact growth and redox metabolism and reveal potential MRS signals for detecting senescent cancer cells in vivo.

Reprogramming of VEGF-mediated extracellular matrix changes through autocrine signaling.

Vascular endothelial growth factor (VEGF) plays key roles in angiogenesis, vasculogenesis, and wound healing. In cancers, including triple negative breast cancer (TNBC), VEGF has been associated with increased invasion and metastasis, processes that require cancer cells to traverse through the extracellular matrix (ECM) and establish angiogenesis at distant sites. To further understand the role of VEGF in modifying the ECM, we characterized VEGF-mediated changes in the ECM of tumors derived from TNBC MDA-MB-231 cells engineered to overexpress VEGF. We established that increased VEGF expression by these cells resulted in tumors with reduced collagen 1 (Col1) fibers, fibronectin, and hyaluronan. Molecular characterization of tumors identified an increase of MMP1, uPAR, and LOX, and a decrease of MMP2, and ADAMTS1. α-SMA, a marker of cancer associated fibroblasts (CAFs), increased, and FAP-α, a marker of a subset of CAFs associated with immune suppression, decreased with VEGF overexpression. Analysis of human data from The Cancer Genome Atlas Program confirmed mRNA differences for several molecules when comparing TNBC with high and low VEGF expression. We additionally characterized enzymatic changes induced by VEGF overexpression in three different cancer cell lines that clearly identified autocrine-mediated changes, specifically uPAR, in these enzymes. Unlike the increase of Col1 fibers and fibronectin mediated by VEGF during wound healing, in the TNBC model, VEGF significantly reduced key protein components of the ECM. These results further expand our understanding of the role of VEGF in cancer progression and identify potential ECM-related targets to disrupt this progression.

Functional roles of FAP-α in metabolism, migration and invasion of human cancer cells.

Fibroblast activation protein-α (FAP-α) is a transmembrane serine protease that is attracting significant interest as it is expressed by a subgroup of cancer-associated fibroblasts that play a role in immune suppression and cancer metastasis. FAP-α is also expressed by some cancer cells, such as melanoma, colorectal and breast cancer cells. Triple negative breast cancer (TNBC) is an aggressive cancer that urgently requires identification of novel targets for therapy. To expand our understanding of the functional roles of FAP-α in TNBC we engineered a human TNBC cell line, MDA-MB-231, to stably overexpress FAP-α and characterized changes in metabolism by 1H magnetic resonance spectroscopy, cell proliferation, migration characterized by wound healing, and invasion. FAP-α overexpression resulted in significant alterations in myoinositol, choline metabolites, creatine, and taurine, as well as a significant increase of migration and invasion, although proliferation remained unaltered. The increase of migration and invasion are consistent with the known activities of FAP-α as an exopeptidase and endopeptidase/gelatinase/collagenase in tissue remodeling and repair, and in cell migration. We additionally determined the effects of FAP-α overexpression on the human fibrosarcoma HT1080 cell line that showed increased migration, accompanied by limited changes in metabolism that identified the dependency of the metabolic changes on cell type. These metabolic data identify a previously unknown role of FAP-α in modifying cancer cell metabolism in the TNBC cell line studied here that may provide new insights into its functional roles in cancer progression.

PD-L1 near Infrared Photoimmunotherapy of Ovarian Cancer Model.

(1) Background: Despite advances in surgical approaches and drug development, ovarian cancer is still a leading cause of death from gynecological malignancies. Patients diagnosed with late-stage disease are treated with aggressive surgical resection and chemotherapy, but recurrence with resistant disease is often observed following treatment. There is a critical need for effective therapy for late-stage ovarian cancer. Photoimmunotherapy (PIT), using an antibody conjugated to a near infrared (NIR) dye, constitutes an effective theranostic strategy to detect and selectively eliminate targeted cell populations. (2) Methods: Here, we are targeting program death ligand 1 (PD-L1) using NIR-PIT in a syngeneic mouse model of ovarian cancer. PD-L1 PIT-mediated cytotoxicity was quantified in RAW264.7 macrophages and ID8-Defb29-VEGF cells in culture, and in vivo with orthotopic ID8-Defb29-VEGF tumors. (3) Results: Treatment efficacy was observed both in vitro and in vivo. (4) Conclusions: Our data highlight the need for further investigations to assess the potential of using NIR-PIT for ovarian cancer therapy to improve the treatment outcome of ovarian cancer.

Hypoxia theranostics of a human prostate cancer xenograft and the resulting effects on the tumor microenvironment.

Hypoxia is frequently observed in human prostate cancer, and is associated with chemoresistance, radioresistance, metastasis, and castrate-resistance. Our purpose in these studies was to perform hypoxia theranostics by combining in vivo hypoxia imaging and hypoxic cancer cell targeting in a human prostate cancer xenograft. This was achieved by engineering PC3 human prostate cancer cells to express luciferase as well as a prodrug enzyme, yeast cytosine deaminase, under control of hypoxic response elements (HREs). Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to HRE in the promoter region of target gene that results in their increased transcription. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation. HRE-driven luciferase expression allowed us to detect hypoxia in vivo to time the administration of the nontoxic prodrug 5-fluorocytosine that was converted by yeast cytosine deaminase, expressed under HRE regulation, to the chemotherapy agent 5-fluorouracil to target hypoxic cells. Conversion of 5-fluorocytosine to 5-fluorouracil was detected in vivo by 19F magnetic resonance spectroscopy. Morphological and immunohistochemical staining and molecular analyses were performed to characterize tumor microenvironment changes in cancer-associated fibroblasts, cell viability, collagen 1 fiber patterns, and HIF-1α. These studies expand our understanding of the effects of eliminating hypoxic cancer cells on the tumor microenvironment and in reducing stromal cell populations such as cancer-associated fibroblasts.

Water and Collagen Content Are High in Pancreatic Cancer: Implications for Quantitative Metabolic Imaging.

In magnetic resonance metabolic imaging, signal from the water content is frequently used for normalization to derive quantitative or semi-quantitative values of metabolites in vivo or ex vivo tumors and tissues. Ex vivo high-resolution metabolic characterization of tumors with magnetic resonance spectroscopy (MRS) provides valuable information that can be used to drive the development of noninvasive MRS biomarkers and to identify metabolic therapeutic targets. Variability in the water content between tumor and normal tissue can result in over or underestimation of metabolite concentrations when assuming a constant water content. Assuming a constant water content can lead to masking of differences between malignant and normal tissues both in vivo and ex vivo. There is a critical need to develop biomarkers to detect pancreatic cancer and to develop novel treatments. Our purpose here was to determine the differences in water content between pancreatic tumors and normal pancreatic tissue as well as other organs to accurately quantify metabolic differences when using the water signal for normalization. Our data identify the importance of factoring the differences in water content between tumors and organs. High-resolution proton spectra of tumors and pancreatic tissue extracts normalized to the water signal, assuming similar water content, did not reflect the significantly increased total choline observed in tumors in vivo without factoring the differences in water content. We identified significant differences in the collagen 1 content between Panc1 and BxPC3 pancreatic tumors and the pancreas that can contribute to the differences in water content that were observed.

HIF and COX-2 expression in triple negative breast cancer cells with hypoxia and 5-fluorouracil.

Our purpose was to understand the effects of normoxia or hypoxia on 5-fluorouracil (5-FU) treatment in triple negative breast cancer (TNBC) cells, and characterize the molecular changes in hypoxia inducible factors (HIFs) and cyclooxygenase-2 (COX-2) following treatment. Cell viability and protein levels of HIFs and COX-2 were determined after wild type and HIF silenced MDA-MB-231 cells, and wild type SUM-149 cells, were treated with 5-FU under normoxia or hypoxia. 5-FU reduced cell viability to the same levels irrespective of normoxia or hypoxia. HIF silenced MDA-MB-231 cells showed comparable changes in cell viability, supporting observations that hypoxia and the HIF pathways did not significantly influence cell viability reduction by 5-FU. Our data suggest that HIF-2α accumulation may predispose cancer cells to cell death under hypoxia. SUM-149 cells that have higher COX-2 and HIF-2α following 24 h of hypoxia, were more sensitive to 96 h of hypoxia compared to MDA-MB-231 cells, and were more sensitive to 5-FU than MDA-MB-231 cells. COX-2 levels changed with hypoxia and with 5-FU treatment but patterns were different between the two cell lines. At 96 h, COX-2 increased in both untreated and 5-FU treated cells under hypoxia in MDA-MB-231 cells. In SUM-149 cells, only treatment with 5-FU increased COX-2 at 96 h of hypoxia. Cells that survive hypoxia and 5-FU treatment may exhibit a more aggressive phenotype. Our results support understanding interactions between HIF and COX-2 with chemotherapeutic agents under normoxia and hypoxia, and investigating the use of COX-2 inhibitors in these settings.

Human Cancer Cell Membrane-Coated Biomimetic Nanoparticles Reduce Fibroblast-Mediated Invasion and Metastasis and Induce T-Cells.

Biomimetic nanoparticles (NPs) combine the flexibility and reproducibility of synthetic materials with the functionality of biological materials. Here, we developed and characterized biomimetic poly(lactic- co-glycolic acid) (PLGA) NPs coated with human cancer cell membrane fractions (CCMFs) to form CCMF-coated PLGA (CCMF-PLGA) NPs. We evaluated the ability of these CCMF-PLGA NPs to disrupt cancer cell-stromal cell interactions and to induce an immune response. Western blot analysis verified the plasma membrane purity of CCMFs. Confocal fluorescence microscopy and flow cytometry confirmed the presence of intact membrane-associated proteins including CXCR4 and CD44 following membrane derivation and coating. CCMFs and CCMF-PLGA NPs were capable of inhibiting cancer cell migration toward human mammary fibroblasts. Intravenous injection of CCMF-PLGA NPs significantly reduced experimental metastasis in vivo. Following immunization of Balb/c mice, near-infrared fluorescence imaging confirmed the migration of NPs to proximal draining lymph nodes (LNs). A higher percentage of CD8+ and CD4+ cytotoxic T-lymphocyte populations was observed in spleens and LNs of CCMF-PLGA NP-immunized mice. Splenocytes isolated from CCMF-PLGA NP-immunized mice had the highest number of interferon gamma-producing T-cells as detected by the ELISpot assay. CCMF-PLGA NPs hold promise for disrupting cancer cell-stromal cell interactions and for priming the immune system in cancer immunotherapy.

MRI and MRS of intact perfused cancer cell metabolism, invasion, and stromal cell interactions.

Because of the spatial and temporal heterogeneities of cancers, technologies to investigate cancer cells and the consequences of their interactions with abnormal physiological environments, such as hypoxia and acidic extracellular pH, with stromal cells, and with the extracellular matrix, under controlled conditions, are valuable to gain insights into the functioning of cancers. These insights can lead to an understanding of why cancers invade and metastasize, and identify effective treatment strategies. Here we have provided an overview of the applications of MRI/MRS/MRSI to investigate intact perfused cancer cells, their metabolism and invasion, and their interactions with stromal cells and the extracellular matrix.

Hypoxia Patterns in Primary and Metastatic Prostate Cancer Environments.

Metastatic dissemination continues to be a major cause of prostate cancer (PCa) mortality, creating a compelling need to understand factors that play a role in the metastatic cascade. Since hypoxia plays an important role in PCa aggressiveness, we characterized patterns of hypoxia in the primary tumor and metastatic environments of a human PCa xenograft. We previously developed and characterized an imaging strategy based on the hypoxia response element (HRE)-driven expression of long-lived enhanced green fluorescent protein (EGFP) and short-lived luciferase (luc) fused to the oxygen-dependent degradation domain in human PCa PC-3 cells. Both reporter proteins were placed under the transcriptional control of a five-tandem repeat HRE sequence. PC-3 cells also constitutively expressed the tdTomato red fluorescent protein, allowing cancer cell detection in vivo. This "timer" strategy can provide information on the temporal evolution of HIF activity and hypoxia in tumors. Here, for the first time, we performed in vivo and ex vivo imaging of this dual HIF reporter system in PC-3 metastatic tumors implanted orthotopically in the prostate and PC-3 nonmetastatic tumors implanted subcutaneously. We observed distinct patterns of EGFP and luc expression in subcutaneous and orthotopic tumors, and in metastatic nodules, that provide new insights into the presence of hypoxia at primary and metastatic tumor sites, and of the role of hypoxia in metastasis.

Molecular causes of elevated phosphoethanolamine in breast and pancreatic cancer cells.

Elevated phosphoethanolamine (PE) is frequently observed in MRS studies of human cancers and xenografts. The role of PE in cell survival and the molecular causes underlying this increase are, however, relatively underexplored. In this study, we investigated the roles of ethanolamine kinases (Etnk-1 and 2) and choline kinases (Chk-α and ß) in contributing to increased PE in human breast and pancreatic cancer cells. We investigated the effect of silencing Etnk-1 and Etnk-2 on cell viability as a potential therapeutic strategy. Both breast and pancreatic cancer cells showed higher PE compared with their nonmalignant counterparts. We identified Etnk-1 as a major cause of the elevated PE levels in these cancer cells, with little or no contribution from Chk-α, Chk-ß, or Etnk-2. The increase of PE observed in pancreatic cancer cells in culture was replicated in the corresponding tumor xenografts. Downregulation of Etnk-1 with siRNA resulted in cell cytotoxicity that correlated with PE levels in breast and pancreatic cancer cells. Etnk-1 may provide a potential therapeutic target in breast and pancreatic cancers.

Metabolic consequences of HIF silencing in a triple negative human breast cancer xenograft.

Hypoxia is frequently encountered in tumors and results in the stabilization of hypoxia inducible factors (HIFs). These factors transcriptionally activate genes that allow cells to adapt to hypoxia. In cancers, hypoxia and HIFs have been associated with increased invasion, metastasis, and resistance to chemo and radiation therapy. Here we have characterized the metabolic consequences of silencing HIF-1α and HIF-2α singly or combined in MDA-MB-231 triple negative human breast cancer xenografts, using non-invasive proton magnetic resonance spectroscopic imaging (1H MRSI) of in vivo tumors, and high-resolution 1H MRS of tumor extracts. Tumors from all three sublines showed a significant reduction of growth rate. We identified new metabolic targets of HIF, and demonstrated the divergent consequences of silencing HIF-1α and HIF-2α individually on some of these targets. These data expand our understanding of the metabolic pathways regulated by HIFs that may provide new insights into the adaptive metabolic response of cancer cells to hypoxia. Such insights may lead to novel metabolism based therapeutic targets for triple negative breast cancer.

Ascites Volumes and the Ovarian Cancer Microenvironment.

Epithelial ovarian cancer is the leading cause of death from gynecologic malignancy among women in developed countries. Epithelial ovarian cancer has a poor prognosis, due to the aggressive characteristics of the disease combined with the lack of effective therapies. Options for late-stage ovarian cancer are limited and invasive, especially once malignant ascites develops. Malignant ascites, a complication observed in terminal ovarian cancer, significantly contributes to poor quality of life and to mortality. Excess accumulation of fluid in the peritoneal cavity occurs due to a combination of impaired fluid drainage and increased net filtration, mostly due to increasing intraperitoneal vascular permeability. Here we applied non-invasive magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) of syngeneic mouse tumors in vivo, and high-resolution 1H MRS of mouse tumor extracts, to characterize the relationship between ascites volumes and the vasculature and metabolism of an experimental model of ovarian cancer. Differences were observed in the tumor vasculature and metabolism in tumors based on ascites volumes that provide new insights into the development of this condition.

Hypoxia Inducible Factors Modify Collagen I Fibers in MDA-MB-231 Triple Negative Breast Cancer Xenografts.

Hypoxia inducible factors (HIFs) are transcription factors that mediate the response of cells to hypoxia. HIFs have wide-ranging effects on metabolism, the tumor microenvironment (TME) and the extracellular matrix (ECM). Here we investigated the silencing effects of two of the three known isoforms, HIF-1α and HIF-2α, on collagen 1 (Col1) fibers, which form a major component of the ECM of tumors. Using a loss-of-function approach for HIF-1α or 2α or both HIF-1α and 2α, we identified a relationship between HIFs and Col1 fibers in MDA-MB-231 tumors. Tumors derived from MDA-MB-231 cells with HIF-1α or 2α or both HIF-1α and 2α silenced contained higher percent fiber volume and lower inter-fiber distance compared to tumors derived from empty vector MDA-MB-231 cells. Depending upon the type of silencing, we observed changes in Col1 degrading enzymes, and enzymes involved in Col1 synthesis and deposition. Additionally, a reduction in lysyl oxidase protein expression in HIF-down-regulated tumors suggests that more non-cross-linked fibers were present. Collectively these results identify the role of HIFs in modifying the ECM and the TME and provide new insights into the effects of hypoxia on the tumor ECM.

Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts.

Cyclooxygenase-2 (COX-2) is a critically important mediator of inflammation that significantly influences tumor angiogenesis, invasion, and metastasis. We investigated the role of COX-2 expressed by triple negative breast cancer cells in altering the structure and function of the extracellular matrix (ECM). COX-2 downregulation effects on ECM structure and function were investigated using magnetic resonance imaging (MRI) and second harmonic generation (SHG) microscopy of tumors derived from triple negative MDA-MB-231 breast cancer cells, and a derived clone stably expressing a short hairpin (shRNA) molecule downregulating COX-2. MRI of albumin-GdDTPA was used to characterize macromolecular fluid transport in vivo and SHG microscopy was used to quantify collagen 1 (Col1) fiber morphology. COX-2 downregulation decreased Col1 fiber density and altered macromolecular fluid transport. Immunohistochemistry identified significantly fewer activated cancer associated fibroblasts (CAFs) in low COX-2 expressing tumors. Metastatic lung nodules established by COX-2 downregulated cells were infrequent, smaller, and contained fewer Col1 fibers.COX-2 overexpression studies were performed with tumors derived from triple negative SUM-149 breast cancer cells lentivirally transduced to overexpress COX-2. SHG microscopy identified significantly higher Col1 fiber density in COX-2 overexpressing tumors with an increase of CAFs. These data expand upon the roles of COX-2 in shaping the structure and function of the ECM in primary and metastatic tumors, and identify the potential role of COX-2 in modifying the number of CAFs in tumors that may have contributed to the altered ECM.

Metabolomic characterization of experimental ovarian cancer ascitic fluid.

INTRODUCTION: Malignant ascites (MA) is a major cause of morbidity that occurs in 37% of ovarian cancer patients. The accumulation of MA in the peritoneal cavity due to cancer results in debilitating symptoms and extremely poor quality of life. There is an urgent unmet need to expand the understanding of MA to design effective treatment strategies, and to improve MA diagnosis. OBJECTIVE: Our purpose here is to contribute to a better characterization of MA metabolic composition in ovarian cancer. METHOD: We determined the metabolic composition of ascitic fluids resulting from orthotopic growth of two ovarian cancer cell lines, the mouse ID8-vascular endothelial growth factor (VEGF)-Defb29 cell line and the human OVCAR3 cell line using high-resolution 1H MRS. ID8-VEGF-Defb29 tumors induce large volumes of ascites, while OVCAR3 tumors induce ascites less frequently and at smaller volumes. To better understand the factors driving the metabolic composition of the fluid, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with 1H NMR. RESULTS: Distinct metabolite patterns were detected in ascitic fluid collected from OVCAR3 and ID8-VEGF-Defb29 tumor bearing mice that were not reflected in the corresponding cell culture or conditioned medium. CONCLUSION: High-resolution 1H NMR metabolic markers of MA can be used to improve characterization and diagnosis of MA. Metabolic characterization of MA can provide new insights into how MA fluid supports cancer cell growth and resistance to treatment, and has the potential to identify metabolic targeting strategies to reduce or eliminate the formation of MA.

Effect of Pantethine on Ovarian Tumor Progression and Choline Metabolism.

Epithelial ovarian cancer remains the leading cause of death from gynecologic malignancy among women in developed countries. New therapeutic strategies evaluated with relevant preclinical models are urgently needed to improve survival rates. Here, we have assessed the effect of pantethine on tumor growth and metabolism using magnetic resonance imaging and high-resolution proton magnetic resonance spectroscopy (MRS) in a model of ovarian cancer. To evaluate treatment strategies, it is important to use models that closely mimic tumor growth in humans. Therefore, we used an orthotopic model of ovarian cancer where a piece of tumor tissue, derived from an ovarian tumor xenograft, is engrafted directly onto the ovary of female mice, to maintain the tumor physiological environment. Treatment with pantethine, the precursor of vitamin B5 and active moiety of coenzyme A, was started when tumors were ~100 mm3 and consisted of a daily i.p. injection of 750 mg/kg in saline. Under these conditions, no side effects were observed. High-resolution 1H MRS was performed on treated and control tumor extracts. A dual-phase extraction method based on methanol/chloroform/water was used to obtain lipid and water-soluble fractions from the tumors. We also investigated effects on metastases and ascites formation. Pantethine treatment resulted in slower tumor progression, decreased levels of phosphocholine and phosphatidylcholine, and reduced metastases and ascites occurrence. In conclusion, pantethine represents a novel potential, well-tolerated, therapeutic tool in patients with ovarian cancer. Further in vivo preclinical studies are needed to confirm the beneficial role of pantethine and to better understand its mechanism of action.

Lymphatic endothelial cells actively regulate prostate cancer cell invasion.

Lymphatic vessels serve as the primary route for metastatic spread to lymph nodes. However, it is not clear how interactions between cancer cells and lymphatic endothelial cells (LECs), especially within hypoxic microenvironments, affect the invasion of cancer cells. Here, using an MR compatible cell perfusion assay, we investigated the role of LEC-prostate cancer (PCa) cell interaction in the invasion and degradation of the extracellular matrix (ECM) by two human PCa cell lines, PC-3 and DU-145, under normoxia and hypoxia, and determined the metabolic changes that occurred under these conditions. We observed a significant increase in the invasion of ECM by invasive PC-3 cells, but not poorly invasive DU-145 cells when human dermal lymphatic microvascular endothelial cells (HMVEC-dlys) were present. Enhanced degradation of ECM by PC-3 cells in the presence of HMVEC-dlys identified interactions between HMVEC-dlys and PCa cells influencing cancer cell invasion. The enhanced ECM degradation was partly attributed to increased MMP-9 enzymatic activity in PC-3 cells when HMVEC-dlys were in close proximity. Significantly higher uPAR and MMP-9 expression levels observed in PC-3 cells compared to DU-145 cells may be one mechanism for increased invasion and degradation of matrigel by these cells irrespective of the presence of HMVEC-dlys. Hypoxia significantly decreased invasion by PC-3 cells, but this decrease was significantly attenuated when HMVEC-dlys were present. Significantly higher phosphocholine was observed in invasive PC-3 cells, while higher glycerophosphocholine was observed in DU-145 cells. These metabolites were not altered in the presence of HMVEC-dlys. Significantly increased lipid levels and lipid droplets were observed in PC-3 and DU-145 cells under hypoxia reflecting an adaptive survival response to oxidative stress. These results suggest that in vivo, invasive cells in or near lymphatic endothelial cells are likely to be more invasive and degrade the ECM to influence the metastatic cascade. Copyright © 2016 John Wiley & Sons, Ltd.

Detection of Pancreatic Cancer-Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance.

The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development.