Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Previous histological investigations of cerebral metastasis demonstrated rapid and drastic changes of the resident glial cells, especially of astrocytes and microglia¹³. To study these changes and interactions with the carcinoma cells, this novel coculture system is well-suited. Other research fields already have long-standing experience with organotypic hippocampal brain slices. One advantage is that the organotypic hippocampal brain slice cultures are viable and effectively preserved for days to weeks, making it suitable for long-term experiments. Since Stoppini's introduction of the organotypic hippocampal slice system in 1991, it has widely been used, for example in research of degenerative diseases. Thus, this innovate coculture system represents a modification of a well-established approach with a range of applications in tumor biology^9,11. The modification offered us a reproducible model to evaluate the grade of tumor invasion afterwards and cultures grown by the interface method are ideally suited for experiments that require a three-dimensional structure. Several techniques have been used to coculture organotypic hippocampal slices with other cells. These include an indirect system between macrophage cells and the organotypic brain slice¹⁴, and a direct coculture of two different slices from the hippocampal region¹⁵. Glioma aggregates have also been cocultured with brain slices¹⁶. These models can be used to analyze cellular and molecular events in the brain slices but do not allow direct, physiological contact between tumor cells, microglia and the brain parenchyma. Furthermore, this method allows the observation of microglia without contamination of bone marrow-derived peripheral monocytes/macrophages. The use of CCR2 and CX3CR1 transgenic mouse model is a critical improvement due to the fact that it is difficult to distinguish the resident microglia from invading monocytes based on their similar properties^17,18,19. Though intracerebral injection of carcinoma cells allows investigating tumor progression, it cannot tell much as to whether the surrounded macrophage-like cells originate from the brain-resident microglia population or from bone marrow-derived peripheral monocytes/macrophages¹⁷. The team of Kettenmann introduced an organotypic brain slice model that involved inoculating glioma cells into brain slices with a micromanipulator ²⁰. However, primary malignant gliomas differ in many regards from metastatic carcinomas. First, malignant gliomas are of mesenchymal origin, do not metastasize outside the nervous system, and migrate/invade as single cells with no border between tumor and brain tissue. By contrast, infiltrative growth is a typical pathological characteristic, and such carcinomas usually migrate/invade as cohorts. Second, carcinomas often try to rebuild epithelial structures in the brain, while glial cells try to separate the tumor from the brain tissue by a (pseudo) capsule. Considering biological and morphological features, malignant glioma and metastasis of carcinomas are not really comparable. For these reasons, we modified and developed a coculture system where we do not inject but coculture a carcinoma cell plug adjacent to the brain slice. Furthermore, we observed microglial and astrocytic accumulation at the border of the tumor plug, meaning that microglia enter the tumor plug and could be easily detected by bright field microscopy and confirmed afterwards by confocal microscopy. The cancer cells invade into the brain slice, which is comparable to the real in vivo situation and to an observation made by Baumert and colleagues, who found an infiltration zone in 63% of autopsy cases with brain metastases²¹.

Because of the missing blood perfusion, there are only resident macrophages/microglia in this culture. Moreover, because of the missing T cells and, therefore, absent allo-reactivity, human carcinoma cells could be used for coculture even with brain slices of immunocompetent mice or rats (NMRI, B6, or Wistar). This could serve as an alternative to the nude mouse model. Since four to five slices can be obtained from each mouse, significantly reduced numbers of animals are required, in comparison to the existing injection models. In addition, the animals do not suffer for a long period of metastatic disease and they do not undergo operative procedures repetitively²².

With this coculture system, we have demonstrated the activation of microglia by cancer cells and the capacity of promoting cancer cell invasion. Additionally, this is the first time, to our knowledge, microglia were found to actively transport carcinoma cells²³.

Despite all these advantages, the coculture system has, indeed, also limitations. It is still an in vitro model missing the steps of metastasis prior to colonization. Because of the lack of perfusion, it is not possible to study the extravasation. Thus, the alternative way to study the extravasation is to use either the in vivo injection model or the modified Boyden chamber system with extracellular matrix, HUVEC and astrocytes to mimic the blood-brain barrier^22,24. The brain slice coculture method is yet a reliable and reproducible model with many advantages and a potential for a wide variety of applications, such as analysis of colonization, in particular with a focus on the role of the resident cells. The combination with other established techniques (such as immunohistochemistry, confocal microscopy and time-lapse microscopy) supports the investigation of direct cell-to-cell interactions. It is an easy alternative and complementation of other techniques and offers access to the investigation of the cues and effects as imposed by the metastatic microenvironment.