Tumor-Endothelial Cell Three-dimensional Spheroids: New Aspects to Enhance Radiation and Drug Therapeutics

Classic cancer research for several decades has focused on understanding the biology of tumor cells in vitro. However, extending these findings to in vivo settings has been impeded owing to limited insights on the impact of microenvironment on tumor cells. We hypothesized that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells, which exist in the tumor microenvironment. To that end, we have developed a system to grow three-dimensional cultures of GFP-4T1 mouse mammary tumor and 2H11 murine endothelial cells in hanging drops of medium in vitro. The presence of 2H11 endothelial cells in these three-dimensional cocultures was found to sensitize 4T1-GFP tumor cells to chemotherapy (Taxol) and, at the same time, protect cells from ionizing radiation. These spheroidal cultures can also be implanted into the dorsal skinfold window chamber of mice for fluorescence imaging of vascularization and disease progression/treatment response. We observed rapid neovascularization of the tumor-endothelial spheroids in comparison to tumor spheroids grown in nude mice. Molecular analysis revealed pronounced up-regulation of several proangiogenic factors in the tumor tissue derived from the tumor-endothelial spheroids compared with tumor-only spheroids. Furthermore, the rate of tumor growth from tumor-endothelial spheroids in mice was faster than the tumor cell-only spheroids, resulting in greater metastasis to the lung. This three-dimensional coculture model presents an improved way to investigate more pertinent aspects of the therapeutic potential for radiation and/or chemotherapy alone and in combination with antiangiogenic agents.     

In Vivo Long-Term Monitoring of Circulating Tumor Cells Fluctuation during Medical Interventions

The goal of this research was to study the long-term impact of medical interventions on circulating tumor cell (CTC) dynamics. We have explored whether tumor compression, punch biopsy or tumor resection cause dissemination of CTCs into peripheral blood circulation using in vivo fluorescent flow cytometry and breast cancer-bearing mouse model inoculated with MDA-MB-231-Luc2-GFP cells in the mammary gland. Two weeks after tumor inoculation, three groups of mice were the subject of the following interventions: (1) tumor compression for 15 minutes using 400 g weight to approximate the pressure during mammography; (2) punch biopsy; or (3) surgery. The CTC dynamics were determined before, during and six weeks after these interventions. An additional group of tumor-bearing mice was used as control and did not receive an intervention. The CTC dynamics in all mice were monitored weekly for eight weeks after tumor inoculation. We determined that tumor compression did not significantly affect CTC dynamics, either during the procedure itself (P = 0.28), or during the 6-week follow-up. In the punch biopsy group, we observed a significant increase in CTC immediately after the biopsy (P = 0.02), and the rate stayed elevated up to six weeks after the procedure in comparison to the tumor control group. The CTCs in the group of mice that received a tumor resection disappeared immediately after the surgery (P = 0.03). However, CTC recurrence in small numbers was detected during six weeks after the surgery. In the future, to prevent these side effects of medical interventions, the defined dynamics of intervention-induced CTCs may be used as a basis for initiation of aggressive anti-CTC therapy at time-points of increasing CTC number.     

Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia

Developed and tested for many years, a variety of tumor hypoxia detection methods have been inconsistent in their ability to predict treatment outcomes or monitor treatment efficacy, limiting their present prognostic capability. These variable results might stem from the fact that these approaches are based on inherently wide-ranging global tumor oxygenation levels based on uncertain influences of necrotic regions present in most solid tumors. Here, we have developed a novel non-invasive and specific method for tumor vessel hypoxia detection, as hypoxemia (vascular hypoxia) has been implicated as a key driver of malignant progression, therapy resistance and metastasis. This method is based on high-frequency ultrasound imaging of α-pimonidazole targeted-microbubbles to the exogenously administered hypoxia marker pimonidazole. The degree of tumor vessel hypoxia was assessed in three mouse models of mammary gland carcinoma (4T1, SCK and MMTV-Wnt-1) and amassed up to 20% of the tumor vasculature. In the 4T1 mammary gland carcinoma model, the signal strength of α-pimonidazole targeted-microbubbles was on average 8-fold fold higher in tumors of pimonidazole-injected mice than in non-pimonidazole injected tumor bearing mice or non-targeted microbubbles in pimonidazole-injected tumor bearing mice. Overall, this provides proof of principle for generating and targeting artificial antigens able to be ‘created’ on-demand under tumor specific microenvironmental conditions, providing translational diagnostic, therapeutic and treatment planning potential in cancer and other hypoxia-associated diseases or conditions.