Ben-Eliyahu, S. | Search bioRxiv

bioRxiv2020

Spontaneous regression of micro-metastases following primary tumor excision: a critical role for primary tumor secretome

Numerous case studies have reported spontaneous regression of recognized metastases following primary tumor (PT) excision, but underlying mechanisms are elusive. Here we present a model of metastases regression and latency following PT excision, and identify potential underlying mechanisms. Using MDA-MB-231HM human breast cancer cells that express highly sensitive luciferase, we were able to monitor early stages of spontaneous metastases development in BALB/c nu/nu mice. Removal of the PT caused marked regression of the smallest micro-metastases, but not of larger metastases, and in vivo supplementation of tumor secretome diminished this regression, suggesting that PT-secreted factors promote early metastatic growth. Correspondingly, cancer cell conditioned medium reduced apoptosis and enhanced MDA-MB-231HM adhesion in vitro. To identify specific mediating factors, cytokine array and proteomic analysis of MDA-MB-231HM secretome were conducted. Results identified significant enrichment of angiogenesis, growth factors binding and activity, focal adhesion, metalloprotease regulation, and apoptosis regulation processes. Simultaneous in vivo blockade of four secreted key potential mediators of these processes, IL-8, PDGFaa, Serpin E1 (PAI-1), and MIF, arrested development of micro-metastases in the presence of the PT. Interestingly, using the public TCGA provisional dataset, high protein levels of these four factors were correlated with poor survival in a cohort of lung adenocarcinoma patients. These results demonstrate regression and latency of micro-metastases following PT excision, and a crucial role for PT-secretome in promoting early metastatic stages in MDA-MB-231HM xenografts. If generalized, such findings can suggest novel approaches to control minimal residual disease during and following PT excision.

cancer biology↗

bioRxiv2019

Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation

Brain metastases are prevalent in various types of cancer, and are often terminal given low efficacy of available therapies. Therefore, preventing them is of outmost clinical relevance and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a TLR9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, NK cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG-C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than non-activated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the anti-tumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia. SummaryBrain metastases are prevalent and often terminal. Thus, reducing their occurrence could markedly improve cancer outcome. We show that systemic prophylactic and perioperative administration of a TLR9 agonist, CpG-C, reduced metastatic growth in experimental and spontaneous brain metastasis models, employing mouse and human tumors. CpG-C was taken up in the brain, without affecting blood-brain barrier integrity and tumor extravasation. In vitro assays, imaging flow cytometry, and intravital imaging pointed to microglia as mediators of CpG-C effects through contact-dependent tumor killing and phagocytosis; corresponding with in vivo mRNA profile. In vivo depletion studies proved that microglia, but not NK cells or monocytes, mediated the beneficial effects of CpG-C; Also hindered by blocking microglial activation. In-toto, perioperative treatment with CpG-C should be considered clinically relevant. SignificancePreventing brain metastases is paramount, as they are considered incurable and their incidence is on the rise due to prolonged survival of cancer patients. Here, we demonstrate that systemic prophylactic treatment with CpG-C reduces peripheral and brain metastasis of mouse and human lung cancers. While traditional therapies are halted during the perioperative period, we found systemic CpG-C treatment during this time frame beneficial in a model of spontaneous brain metastases following excision of a primary melanoma tumor, comprehensively mimicking the clinical setting. Mechanistically, we show microglia activation with CpG-C results in tumor cell eradication, pointing to microglia as potential therapeutic targets. Importantly, CpG-ODNs have negligible toxicity in humans. Therefore, CpG-C may be used prophylactically and during the perioperative period in high-risk cancers.

neuroscience↗