Customized Image Analysis Tools/Modules
Our lab specializes in immunohistochemical and immunofluorescent pathology to resolve biological questions on microenvironmental cross-talk among diverse populations of neoplastic and non-neoplastic cells. We first ask an intriguing research question; then, we formulate our hypothesis; then, we develop and customize image analysis tools/modules that are uniquely tied to the nature of this hypothesis. In this section, we describe selected examples of our most interesting image analysis tools/modules that are publicly available.
Blood Vessel Permeability (Associated with Cancer Cell Intravasation, a.k.a. TMEM-Permeability)
This assay takes advantage of the intravenous injection of high-molecular weight dextran (155 kDa-dextran) in mice. Typically, 155 kDa-Dextran is restricted and confined within the blood vessel wall. However, it can cross the endothelium barrier under conditions, in which the endothelial tight junctions are temporarily dissolved. This very frequently happens intratumorally due to the increased activity of TMEM doorways, specialized niches within the tumor microenvironment capable of pumping disseminating tumor cells into the blood vasculature. The abrupt and massive breaking of endothelial junctions at TMEM doorways, followed by the leakage of 155 kDa-Dextran into the tumor tissue, can then be visualized using multichannel immunofluorescence in fixed tissues, as a "bursting" phenomenon. You may read the full protocol of how to perform this assay here.
Multichannel immunofluorescence of Endomucin (green; first column), 155-kDa dextran-TMR (red; second column), their merged image along with DAPI (third column), the thresholded blood vessel and extravascular dextran masks (fourth column), and the corresponding sequential section of TMEM IHC (fifth column) in MMTV-PyMT mice. Top: Vascular profile away from TMEM, appearing as a "non-leaky" vascular profile. Bottom: TMEM-associated vascular profile, appearing as a "leaky" vascular profile.