Non-reversible tissue fixation retains extracellular vesicles for in situ imaging

Summary Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.

The 4T1 mammary carcinoma is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone The 4T1 tumor has several characteristics that make it a suitable experimental animal model for human mammary cancer. First, tumor cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site, as described in this unit. Second, as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor. Also, the progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer. In this unit, a protocol describes surgical removal of the primary tumor, so that metastatic disease can be studied in an animal setting comparable to the clinical situation where the primary tumor is surgically removed, and metastatic foci remain intact. Another advantage of 4T1 is its resistance to 6-thioguanine. This property enables precise quantitation of metastatic cells, even when they are disseminated and at sub-microscopic levels in distant organs, as described here.