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Colorectal cancer (CRC) raises considerable clinical challenges, including a high mortality rate once the tumor spreads to distant sites. At this advanced stage, more accurate prediction of prognosis and treatment outcome is urgently needed. The role of cancer immunity in metastatic CRC (mCRC) is poorly understood. Here, we explore cellular immune cell status in patients with multi-organ mCRC. We analyzed T cell infiltration in primary tumor sections, surveyed the lymphocytic landscape of liver metastases, and assessed circulating mononuclear immune cells. Besides asking whether immune cells are associated with survival at this stage of the disease, we investigated correlations between the different tissue types; as this could indicate a dominant immune phenotype. Taken together, our analyses corroborate previous observations that higher levels of CD8+ T lymphocytes link to better survival outcomes. Our findings therefore extend evidence from earlier stages of CRC to indicate an important role for cancer immunity in disease control even after metastatic spreading to multiple organs. This finding may help to improve predicting outcome of patients with mCRC and suggests a future role for immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

Abstract Background Plexin D1 is expressed on both tumor-associated endothelium and malignant cells in a number of clinical brain tumors. Recently we demonstrated that Plexin D1 expression is correlated with tumor invasion level and metastasis in a human melanoma progression series. The objective of this study was to examine whether Plexin D1 might be clinically useful as a pan-tumor vessel and pan-tumor cell target in solid tumors. Methods We examined Plexin D1 expression in clinical solid tumors (n = 77) of different origin, a selection of pre-malignant lesions (n = 29) and a variety of non-tumor related tissues (n = 52) by immunohistochemistry. Signals were verified in a selection of tissues via mRNA in situ hybridization. Results Plexin D1 is abundantly expressed on both activated established tumor vasculature and malignant cells in the majority of primary and metastatic clinical tumors, as well as on macrophages and fibroblasts. Importantly, in non-tumor related tissues Plexin D1 expression is restricted to a subset of, presumably activated, fibroblasts and macrophages. Conclusion We demonstrate that Plexin D1 is in general ubiquitously expressed in tumor but not normal vasculature, as well as in malignant cells in a wide range of human tissues. This expression profile highlights Plexin D1 as a potentially valuable therapeutic target in clinical solid tumors, enabling simultaneous targeting of different tumor compartments.

Tissue specimens taken from primary tumors or metastases contain important information for diagnosis and treatment of cancer patients. Multiplex imaging allows in situ visualization of heterogeneous cell populations, such as immune cells, in tissue samples. Most image processing pipelines first segment cell boundaries and then measure marker expression to assign cell phenotypes. In dense tissue environments, this segmentation-first approach can be inaccurate due to segmentation errors or overlapping cells. Here, we introduce the machine-learning pipeline "ImmuNet", which identifies positions and phenotypes of cells without segmenting them. ImmuNet is easy to train: human annotators only need to click on an immune cell and score its expression of each marker-drawing a full cell outline is not required. We trained and evaluated ImmuNet on multiplex images from human tonsil, lung cancer, prostate cancer, melanoma, and bladder cancer tissue samples and found it to consistently achieve error rates below 5%-10% across tissue types, cell types, and tissue densities, outperforming a segmentation-based baseline method. Furthermore, we externally validate ImmuNet results by comparing them to flow cytometric cell count measurements from the same tissue. In summary, ImmuNet is an effective, simpler alternative to segmentation-based approaches when only cell positions and phenotypes, but not their shapes, are required for downstream analyses. Thus, ImmuNet helps researchers to analyze cell positions in multiplex tissue images more easily and accurately., (© The Author(s) 2024. Published by Oxford University Press.)

We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood supply (B. Kusters et al., Cancer Res., 62: 341-345, 2002). Here, we compare the activities of the 121, 165, and 189 VEGF-A isoforms in this model by transfecting Mel57 cells with the respective cDNAs and by injecting the resulting stably transfected cell lines in the internal carotid arteries of nude mice (n = 10 for each isoform). Although the three isoforms had similar potency to induce endothelial cell proliferation, VEGF(121) expression did not result in sprouting angiogenesis but rather led to extensive vasodilation and increased permeability of pre-existing, predominantly peritumoral vessels. Sometimes, proliferating endothelial cells accumulated in vessel lumina, giving these a microvascular, glomeruloid, proliferation-like appearance. Expression of VEGF(165) or VEGF(189) was associated with induction of an intratumoral neovascular bed. In VEGF(165)-expressing tumors, daughter endothelial cells were distributed among newly formed vessels that were extensively dilated. This also occurred in VEGF(189) tumors, but there, vasodilation was less pronounced. Using contrast-enhanced magnetic resonance imaging, the different vascular phenotypes were visualized on characteristic radiological images. VEGF(165) expression was the most unfavorable of the three. Mice carrying VEGF(165) tumors became moribund earlier than those carrying VEGF(121)-expressing tumors (16 +/- 4 days versus 22 +/- 3 days). Our data demonstrate that VEGF-A isoforms differ in angiogenic properties that can be visualized by contrast-enhanced magnetic resonance imaging.

We investigated the mechanisms of vascularization in a brain metastases model of malignant melanoma. Parenchymal metastases expressing little vascular endothelial growth factor-A (VEGF-A) co-opted the preexistent brain vasculature, leading to an infiltrative phenotype. Metastases of the human melanoma cell line Mel57, engineered to express recombinant VEGF-A(165), showed accelerated growth in a combined expansive and infiltrative pattern with marked central necrosis. This difference in growth profile was accompanied by dilation of co-opted intra- and peritumoral vessels with concomitant induction of vascular permeability. Our data show that modulation of preexistent vasculature can contribute to malignant progression without induction of sprouting angiogenesis.