Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.
Pancreatic ductal adenocarcinomas (PDAC) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC- derived exosomes induce liver pre-metastatic niche formation in naïve mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared to patients whose pancreatic tumors did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.
Highlights d Proteomic profiles of extracellular vesicles and particles (EVPs) from 426 human samples d Identification of pan-EVP markers d Characterization of tumor-derived EVP markers in human tissues and plasma d EVP proteins can be useful for cancer detection and determining cancer type
AUTHOR CONTRIBUTIONS G.R. designed the experimental approach, performed the experimental work, analyzed the data, coordinated the project and wrote the manuscript. A.H. performed primary tumour growth and exosome education in vivo studies, cancer cell proliferation in vitro studies, cancer cell culture and exosome isolation, coordinated the project and wrote the manuscript. C.M.K. generated CEMIP overexpression, performed molecular cloning work and genetic manipulation of cancer cells, cancer cell culture and exosome isolation, coordinated the project and wrote the manuscript. I.R.M. performed brain slice ex vivo FACS analysis and exosome education in vivo studies, cancer cell culture and exosome isolation, coordinated the project, wrote and reviewed the manuscript. L.S. performed brain slice ex vivo experimental work, tissue processing and immunostaining, ex vivo and in vivo ImageJ data analysis and quantification, cancer cell invasion in vitro studies, western blot analysis, cancer cell culture and exosome isolation, and contributed to figure panel assembly. D.F. performed density gradient exosome isolation, characterization and analysis, western blot analysis, and cancer cell culture. H.S.K. and P.R.O. performed RNA sequencing data analysis. I.S. performed tissue processing and immunostaining, ex vivo and in vivo ImageJ data analysis and quantification, cancer cell culture and exosome isolation. I.C.S. performed western blot analysis and assisted in analysis of human data.
Dendritic cells (DCs) are key regulators of host immunity that are capable of inducing either immune tolerance or activation. In addition to their well-characterized role in shaping immune responses to foreign pathogens, DCs are also known to be critical for the induction and maintenance of anti-tumor immune responses. Therefore, it is important to understand how tumors influence the function of DCs and the quality of immune responses they elicit. Although the majority of studies in this field to date have utilized either immortalized DC lines or DC populations that have been generated under artificial conditions from hematopoietic precursors in vitro, we wished to investigate how tumors impact the function of already differentiated, tissue-resident DCs. Therefore, we used both an ex vivo and in vivo model system to assess the influence of melanoma-derived factors on DC maturation and activation. In ex vivo studies with freshly isolated splenic DCs, we demonstrate that the extent to which DC maturation and activation are altered by these factors correlates with melanoma tumorigenicity, and we identify partial roles for tumor-derived transforming growth factor (TGF)β1 and vascular endothelial growth factor (VEGF)-A in the altered functionality of DCs. In vivo studies using a lung metastasis model of melanoma also demonstrate tumorigenicity-dependent alterations to the function of lung-resident DCs, and skewed production of proinflammatory cytokines and chemokines by these tumor-altered cells is associated with recruitment of an immune infiltrate that may ultimately favor tumor immune escape and outgrowth.
Dendritic cells function as potent regulators of both innate and adaptive immunity to tumors and the regulatory activities of these cells are tightly linked to their maturation and activation status. Despite the critical role played by dendritic cells in the induction of anti-tumor immune responses, the number of dendritic cells that can be isolated from experimental animals is limiting and often precludes in-depth analyses of these cells. To overcome this limitation, dendritic cell lines have been established and have facilitated the experimental study of dendritic cell biology. In this study we compare the dendritic cell lines DC2.4 and JAWSII as in vitro model systems for studying the influence of melanoma-derived factors on dendritic cell maturation and activation. Using flow cytometry and ELISA analyses, we evaluate the expression of costimulatory/MHC class II molecules and proinflammatory cytokines/chemokines by these dendritic cell lines in their resting state and following LPS stimulation in the presence or absence of B16-F1 melanoma-derived factors. Results: We demonstrate that soluble B16-F1-derived factors suppress the LPSinduced upregulation of CD40, CD80, CD86 and MHC class II on both the DC2.4 and JAWSII dendritic cell lines. Interestingly, LPS-induced secretion by DC2.4 cells of the proinflammatory cytokines/chemokines TNF-α, IP-10, MIP-1α, MIP-1β and MCP-1 is also altered by B16-F1-derived factors, whereas JAWSII cell cytokine/chemokine production is affected to a lesser extent by such factors, with only IL-1β and IP-10 production being suppressed. Conclusions/Recommendations: We conclude that melanoma-derived factors can suppress dendritic cell maturation/activation and that the DC2.4 and JAWSII dendritic cell lines are effective in vitro models for future studies that aim to (1) identify factors that influence both the susceptibility and the resistance of dendritic cells to tumor-mediated immunosuppression and (2) investigate the influence of tumor-altered dendritic cells on the quality of anti-tumor T cell responses.
Angiogenesis and the establishment of a vascular network by tumor‐associated endothelial cells (ECs) are well‐described in cancer. Although ECs play an essential role in tumorigenesis by constructing vasculature, other mechanisms through which ECs support tumor survival and metastasis is largely unknown. Since Notch signaling is critical for breast carcinogenesis, we investigated the potential role of ECs in mediating Notch activation. We propose that by upregulating their expression of Notch ligand Jagged 1(Jag 1), ECs induce aberrant paracrine Notch signaling in breast cancer cells, thereby providing an instructive signal for tumor growth and progression. To determine the role of EC Jag 1 expression on breast carcinogenesis, we established 3D co‐cultures of human breast cancer cells (MDA‐MB‐231) with either wild‐type or Jag 1 knockdown (KD) human umbilical vein ECs (HUVECs). We found that Jag1 loss in ECs induced decreased tumor growth, mammo‐angioshpere formation, and CD40 and CD133 expression in MDA‐231 cells. Co‐injection of Jag1 KD co‐cultures into immunocompromised mice also resulted in decreased tumor weight and volume compared to wild type co‐injections. Conditional Jag1 deletion in ECs of PyMT‐MMTV transgenic mice also led to decreased primary tumor growth and lung metastasis. These results suggest that EC Jag1 upregulation supports Notch‐dependent breast cancer cell growth and metastasis Grant Funding Source: Supported by the Howard Hughes Medical Institute
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