Grace Maresh

The emerging roles of extracellular vesicles as communication vehicles within the tumor microenvironment and beyond

Tumors evolve in complex and dynamic microenvironments that they rely on for sustained growth, invasion, and metastasis. Within this space, tumor cells and non-malignant cells are in frequent communication. One specific mode of communication that has gained recent attention is the release of extracellular vesicles (EVs). EVs are lipid bilayer-bound vehicles that are released from the cell membrane and carry nucleic acids, proteins, and lipids to neighboring or distant cells. EVs have been demonstrated to influence a multitude of processes that aid in tumor progression including cellular proliferation, angiogenesis, migration, invasion, metastasis, immunoediting, and drug resistance. The ubiquitous involvement of EVs on cancer progression makes them very suitable targets for novel therapeutics. Furthermore, they are being studied as specific markers for cancer diagnostics, prognosis, and even as chemotherapy drug-delivery systems. This review focuses on the most recent advances in EV knowledge, some current and potential problems with their use, and some proposed solutions to consider for the future.

A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis

High-grade urothelial cell carcinoma of the bladder has a poor prognosis when lymph nodes are involved. Despite curative therapy for clinically-localized disease, over half of the muscle-invasive urothelial cell carcinoma patients will develop metastases and die within 5 years. There are currently no described xenograft models that consistently mimic urothelial cell carcinoma metastasis. To develop a patient-derived orthotopic xenograft model to mimic clinical urothelial cell carcinoma progression to metastatic disease, the urothelial cell carcinoma cell line UM-UC-3 and two urothelial cell carcinoma patient specimens were doubly tagged with Luciferase/RFP and were intra-vesically (IB) instilled into NOD/SCID mice with or without lymph node stromal cells (HK cells). Mice were monitored weekly with bioluminescence imaging to assess tumor growth and metastasis. Primary tumors and organs were harvested for bioluminescence imaging, weight, and formalin-fixed for hematoxylin and eosin and immunohistochemistry staining. In this patient-derived orthotopic xenograft model, xenograft tumors showed better implantation rates than currently reported using other models. Xenograft tumors histologically resembled pre-implanted primary specimens from patients, presenting muscle-invasive growth patterns. In the presence of HK cells, tumor formation, tumor angiogenesis, and distant organ metastasis were significantly enhanced in both UM-UC-3 cells and patient-derived specimens. Thus, we established a unique, reproducible patient-derived orthotopic xenograft model using human high-grade urothelial cell carcinoma cells and lymph node stromal cells. It allows for investigating the mechanism involved in tumor formation and metastasis, and therefore it is useful for future testing the optimal sequence of conventional drugs or the efficacy of novel therapeutic drugs.

Abstract A11: Using a patient-derived orthotopic xenograft (PDOX) model to screen drugs targeting renal cell carcinoma (RCC) metastasis: A personalized therapeutic strategy

Renal cell carcinoma (RCC) is the most common solid tumor of the adult kidney, with a mortality of 30-40%(1). RCC is highly metastatic and potentially lethal. 25-30% of RCC patients have metastatic disease at diagnosis, and 40-50% of patients develop metastases later. The 5-year survival for metastatic RCC patients is less than 10%(2), due to its highly-resistant to current therapies. The challenge of managing RCC patients in the clinic often involves trying different drugs one after another until one works, so individualized therapy is urgently needed. Our objective was to establish a patient-derived orthotopic xenograft (PDOX) intra-renal sub-capsular injection NOD/SCID mouse model that recapitulates metastatic RCC for personalized therapeutic strategies. Two luciferase-tagged RCC cell lines (A498-Luc, 769P-Luc) were used to determine the IC50 of sunitinib, pazopanib, and everolimus using bioluminescent imaging (BLI) of in vitro cell viability assay at 48 hrs. A consented human RCC specimen (KiCa-Pt58) was obtained via radical nephrectomy and tumor cells tagged with luciferase. KiCa-Pt58-Luc cells (0.01 million) were then injected sub-capsularly into the left kidneys of NOD/SCID mice. Mice bearing kidney tumors were split into 1 control and 4 sequential treatment groups receiving 40mg/kg sunitinib, 40mg/kg pazopanib, or 5mg/kg everolimus three times per week via gavage. Tumor growth was monitored weekly by BLI. Drug responses were evaluated based on BLI data and decision made for whether to switch to the next sequence drug. At necropsy, primary tumors and mouse lungs were collected for BLI, weights, and histology. The IC50 was 6μM for sunitinib and 3.6μg/ml for pazopanib. In our PDOX model, patient tumors successfully produced primary tumor and spontaneous lung metastasis as seen in RCC patients (45/45). Selected drug dosages delivered by gavage were safe, effective and without adverse effects. Drug sequence of pazopanib switch to everolimus is the best among 4 combinations tested for primary tumor progress and lung metastasis for patient tumor KiCa-Pt58. Our unique PDOX model provides a platform for testing RCC drugs simultaneously for responses in primary tumor progression and metastasis. It could lead to the development of realistic, durable, and individualized treatments for RCC patients. Citation Format: Jessie Gills, Ravan Moret, Xin Zhang, Ashley Richman, John Nelson, Grace Maresh, Christudas Morais, Glenda Gobe, Sunil Talwar, Marc Matrana, Stephen Bardot, Li Li. Using a patient-derived orthotopic xenograft (PDOX) model to screen drugs targeting renal cell carcinoma (RCC) metastasis: A personalized therapeutic strategy. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A11.

Abstract 1558: The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis

Introduction: Metastatic disease is responsible for 90% of colorectal cancer (CRC) deaths. Studies suggest that metastasis is closely associated with the presence of CRC tumor-initiating cells (Co-TIC) and their interaction with the lymph node (LN) stromal microenvironment. Prior to developing extra-nodal metastasis, these cells acquire a chemotherapy-resistant phenotype developing genetic alterations making them resistant to conventional treatments. In addition to cell-cell contact and secreted molecules, a recently discovered means of intercellular signaling is the exchange of extra-cellular vesicles. These microvesicles (MVs) carry complex biological information, including mRNA, miRNA, as well as soluble and transmembrane proteins that can affect the behavior of target cells. MVs have been detected in patient specimens with diverse malignancies and may play a role in communication between the LN stromal microenvironment and Co-TIC. We hypothesize that MVs are involved in intracellular trafficking between LN stromal cells and CRC cells promoting tumor formation and distant organ metastasis. Methods: MVs released by human mesenteric LN stromal cells (LNSC) derived from surgical specimens and the established LN stromal cell line (HK cell) were isolated using differential centrifugation and gradient purification. The MVs were visualized using GFP-HK cell and RFP-HT-29 cell (CRC cell line) and florescence microscopy. The functional properties of LN stromal MVs and their effect on CRC proliferation and metastasis was analyzed using established in vitro co-culture models and a humanized orthotopic intra-rectal (IR) injection mouse model, tracked by bioluminescent imaging (BLI). Results: A 100,000 g pellet containing MVs derived from LNSC and HK cells have a similar size profile when analyzed by NanoSight. Budding CD63-RFP tagged MVs were released by LNSC and HK cells and uptake by GFP tagged CRC cells was confirmed through time-lapse experiments using deconvoluting microscopy. When HK cell or LN stromal cell-derived MVs were co-cultured with HT-29 cells in vitro, they supported HT-29 cell growth at a similar level as that of HK cell or LN stromal cell conditioned media, respectively. By adding LNSC- or HK-derived MVs to HT-29-Luc cells or patient derived CRC cells (CRC-Pt-Luc cells) in our IR model, we demonstrated that MVs enhanced CRC tumor growth as well as distant organ metastasis in vivo. Conclusion: MVs isolated from LNSCs traffic between the stromal cells and CRC cells. These MVs promote tumor formation and distant organ metastasis in vivo suggesting that they play a crucial role in the communication between the LN stromal microenvironment and CRC cells. Further analyzing the functional properties of effector MV RNAs may help identify novel targetable candidates for therapeutic strategies that target CRC metastasis using our unique patient derived orthotopic mouse model. Citation Format: Xin Zhang, Ryan Sullivan, Nathan Hite, Grace Maresh, Linh Hellmers, Zhen Lin, Erik Flemington, Carlos Salomon, Heather Green, David Margolin, Li Li. The critical role of lymph node stromal cell-derived microvesicles in colorectal cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1558.