Splenectomy reduces lung metastases and tumoral and metastatic niche inflammation.

The immune microenvironment plays a crucial role in supporting tumor growth and metastasis. Tumor-associated macrophages (TAMs) and neutrophils (TANs) are essential components of this microenvironment and affect tumor growth and progression in almost all solid neoplasms. Furthermore, TAMs, TANs and tumor-infiltrating dendritic cells (TIDCs) are found to infiltrate specific distant organs to prepare them as a site for metastatic cell seeding, forming the pre-metastatic niche. The spleen was identified as a major reservoir and source of circulating and tumor infiltrating immune cells. However, discrepancies about its role in supporting tumor growth exist. Thus, here we investigated the role of splenectomy in primary tumor and metastatic growth, and in the formation of an inflammatory niche. In a murine 4T1 and E0771 breast and Panc02 pancreatic cancer model, our results show that while splenectomy reduces the number of infiltrating TAMs, TANs and TIDCs within primary tumors, it does not affect its growth. In line, fewer TAMs, TANs and TIDCs accumulate in the metastatic microenvironment after splenectomy. Interestingly though, this affected metastatic growth depending on the metastatic route/site. The number of hematogenous breast cancer lung metastases was reduced after splenectomy but no effect was observed in breast or pancreatic lymph node metastases. Moreover, we observed that the immune composition of the pre-metastatic niche in lungs of breast cancer bearing mice was altered, and that this could cause the reduction of metastases. Altogether, our results highlight that splenectomy affects the immune microenvironment not only of primary tumors but also of pre-metastatic and metastatic sites.

Role of retinal pigment epithelium-derived exosomes and autophagy in new blood vessel formation.

Autophagy and exosome secretion play important roles in a variety of physiological and disease states, including the development of age-related macular degeneration. Previous studies have demonstrated that these cellular mechanisms share common pathways of activation. Low oxidative damage in ARPE-19 cells, alters both autophagy and exosome biogenesis. Moreover, oxidative stress modifies the protein and genetic cargo of exosomes, possibly affecting the fate of surrounding cells. In order to understand the connection between these two mechanisms and their impact on angiogenesis, stressed ARPE-19 cells were treated with a siRNA-targeting Atg7, a key protein for the formation of autophagosomes. Subsequently, we observed the formation of multivesicular bodies and the release of exosomes. Released exosomes contained VEGFR2 as part of their cargo. This receptor for VEGF-which is critical for the development of new blood vessels-was higher in exosome populations released from stressed ARPE-19. While stressed exosomes enhanced tube formation, exosomes became ineffective after silencing VEGFR2 in ARPE-19 cells and were, consequently, unable to influence angiogenesis. Moreover, vessel sprouting in the presence of stressed exosomes seems to follow a VEGF-independent pathway. We propose that abnormal vessel growth correlates with VEGFR2-expressing exosomes release from stressed ARPE-19 cells, and is directly linked to autophagy.

Metastasis-associated fibroblasts promote angiogenesis in metastasized pancreatic cancer via the CXCL8 and the CCL2 axes.

The characteristic desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) is a key contributor to its lethality. This stromal microenvironment is populated by cancer-associated fibroblasts (CAFs) that interact with cancer cells to drive progression and chemo-resistance. Research has focused on CAFs in the primary tumour but not in metastases, calling into question the role of analogous metastasis-associated fibroblasts (MAFs). We infer a role of MAFs in murine hepatic metastases following untargeted treatment with the anti-angiogenic drug sunitinib in vivo. Treated metastases were smaller and had fewer stromal cells, but were able to maintain angiogenesis and metastasis formation in the liver. Furthermore, sunitinib was ineffective at reducing MAFs alongside other stromal cells. We speculate that cancer cells interact with MAFs to maintain angiogenesis and tumour progression. Thus, we tested interactions between metastatic pancreatic cancer cells and fibroblasts using in vitro co-culture systems. Co-cultures enhanced fibroblast proliferation and induced angiogenesis. We identify carcinoma-educated fibroblasts as the source of angiogenesis via secretions of CXCL8 (aka IL-8) and CCL2 (aka MCP-1). Overall, we demonstrate that metastasis-associated fibroblasts have potential as a therapeutic target and highlight the CXCL8 and CCL2 axes for further investigation.

Reduction of Liver Metastasis Stiffness Improves Response to Bevacizumab in Metastatic Colorectal Cancer.

Tumors are influenced by the mechanical properties of their microenvironment. Using patient samples and atomic force microscopy, we found that tissue stiffness is higher in liver metastases than in primary colorectal tumors. Highly activated metastasis-associated fibroblasts increase tissue stiffness, which enhances angiogenesis and anti-angiogenic therapy resistance. Drugs targeting the renin-angiotensin system, normally prescribed to treat hypertension, inhibit fibroblast contraction and extracellular matrix deposition, thereby reducing liver metastases stiffening and increasing the anti-angiogenic effects of bevacizumab. Patients treated with bevacizumab showed prolonged survival when concomitantly treated with renin-angiotensin inhibitors, highlighting the importance of modulating the mechanical microenvironment for therapeutic regimens.

VEGFR1+ Metastasis-Associated Macrophages Contribute to Metastatic Angiogenesis and Influence Colorectal Cancer Patient Outcome.

PURPOSE: To investigate the clinical relevance of macrophages in liver metastasis of colorectal cancer and their influence on angiogenesis and patient survival. Moreover to evaluate specific blood monocytes as markers of disease recurrence.Experimental design: In a mouse model with spontaneous liver metastasis, the angiogenic characteristics of tumor- and metastasis (MAM)-associated macrophages were evaluated. Macrophages and the vasculature from 130 primary tumor (pTU) and 123 patients with liver metastasis were assessed. In vivo and in human samples, the clinical relevance of macrophage VEGFR1 expression was analyzed. Blood samples from patients (n = 157, 80 pTU and 77 liver metastasis) were analyzed for assessing VEGFR1-positive (VEGFR1+) cells as suitable biomarkers of disease recurrence. RESULTS: The number of macrophages positively correlated with vascularization in metastasis. Both in the murine model as well as in primary isolated human cells, a subpopulation of MAMs expressing VEGFR1 were found highly angiogenic. While VEGFR1 expression in pTU patients did not predict prognosis; high percentage of VEGFR1+ cells in liver metastasis was associated with worse patient outcome. Interestingly, VEGFR1+-circulating monocytes in blood samples from patients with liver metastasis not only predicted progression but also site of recurrence. CONCLUSIONS: Our findings identify a new subset of proangiogenic VEGFR1+ MAMs in colorectal cancer that support metastatic growth and may become a liquid biomarker to predict disease recurrence in the liver.

Caspase-8 modulates physiological and pathological angiogenesis during retina development.

During developmental angiogenesis, blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether, how, cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates cell death via both apoptosis and necroptosis. Here, we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal retina angiogenesis. EC-specific Casp-8-KO pups (Casp-8ECKO) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting, and migration independently of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 MAPK downstream of receptor-interacting serine/threonine protein kinase 3 (RIPK3) and destabilization of vascular endothelial cadherin (VE-cadherin) at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR) resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-8ECKO pups. Taking these data together, we show that Casp-8 acts in a cell death-independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP.

YAP/TAZ Orchestrate VEGF Signaling during Developmental Angiogenesis.

Vascular endothelial growth factor (VEGF) is a major driver of blood vessel formation. However, the signal transduction pathways culminating in the biological consequences of VEGF signaling are only partially understood. Here, we show that the Hippo pathway effectors YAP and TAZ work as crucial signal transducers to mediate VEGF-VEGFR2 signaling during angiogenesis. We demonstrate that YAP/TAZ are essential for vascular development as endothelium-specific deletion of YAP/TAZ leads to impaired vascularization and embryonic lethality. Mechanistically, we show that VEGF activates YAP/TAZ via its effects on actin cytoskeleton and that activated YAP/TAZ induce a transcriptional program to further control cytoskeleton dynamics and thus establish a feedforward loop that ensures a proper angiogenic response. Lack of YAP/TAZ also results in altered cellular distribution of VEGFR2 due to trafficking defects from the Golgi apparatus to the plasma membrane. Altogether, our study identifies YAP/TAZ as central mediators of VEGF signaling and therefore as important regulators of angiogenesis.