Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis.

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.

EPHA2-dependent outcompetition of KRASG12D mutant cells by wild-type neighbors in the adult pancreas.

As we age, our tissues are repeatedly challenged by mutational insult, yet cancer occurrence is a relatively rare event. Cells carrying cancer-causing genetic mutations compete with normal neighbors for space and survival in tissues. However, the mechanisms underlying mutant-normal competition in adult tissues and the relevance of this process to cancer remain incompletely understood. Here, we investigate how the adult pancreas maintains tissue health in vivo following sporadic expression of oncogenic Kras (KrasG12D), the key driver mutation in human pancreatic cancer. We find that when present in tissues in low numbers, KrasG12D mutant cells are outcompeted and cleared from exocrine and endocrine compartments in vivo. Using quantitative 3D tissue imaging, we show that before being cleared, KrasG12D cells lose cell volume, pack into round clusters, and E-cadherin-based cell-cell adhesions decrease at boundaries with normal neighbors. We identify EphA2 receptor as an essential signal in the clearance of KrasG12D cells from exocrine and endocrine tissues in vivo. In the absence of functional EphA2, KrasG12D cells do not alter cell volume or shape, E-cadherin-based cell-cell adhesions increase and KrasG12D cells are retained in tissues. The retention of KRasG12D cells leads to the early appearance of premalignant pancreatic intraepithelial neoplasia (PanINs) in tissues. Our data show that adult pancreas tissues remodel to clear KrasG12D cells and maintain tissue health. This study provides evidence to support a conserved functional role of EphA2 in Ras-driven cell competition in epithelial tissues and suggests that EphA2 is a novel tumor suppressor in pancreatic cancer.

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency.

BACKGROUND: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. METHODS: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. FINDINGS: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. CONCLUSIONS: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. FUNDING: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).

L-Selectin Enhanced T Cells Improve the Efficacy of Cancer Immunotherapy.

The homing molecule, L-selectin (CD62L), is commonly used as a T cell activation marker, since expression is downregulated following engagement of the T cell receptor. Studies in mice have shown that CD62L+ central memory T cells are better at controlling tumor growth than CD62L- effector memory T cells, while L-selectin knockout T cells are poor at controlling tumor growth. Here, we test the hypothesis that T cells expressing genetically modified forms of L-selectin that are maintained following T cell activation (L-selectin enhanced T cells) are better at controlling tumor growth than wild type T cells. Using mouse models of adoptive cell therapy, we show that L-selectin enhancement improves the efficacy of CD8+ T cells in controlling solid and disseminated tumor growth. L-selectin knockout T cells had no effect. Checkpoint blockade inhibitors synergized with wild type and L-selectin enhanced T cells but had no effect in the absence of T cell transfers. Reduced tumor growth by L-selectin enhanced T cells correlated with increased frequency of CD8+ tumor infiltrating T cells 21 days after commencing therapy. Longitudinal tracking of Zirconium-89 (89Zr) labeled T cells using PET-CT showed that transferred T cells localize to tumors within 1 h and accumulate over the following 7 days. L-selectin did not promote T cell homing to tumors within 18 h of transfer, however the early activation marker CD69 was upregulated on L-selectin positive but not L-selectin knockout T cells. L-selectin positive and L-selectin knockout T cells homed equally well to tumor-draining lymph nodes and spleens. CD69 expression was upregulated on both L-selectin positive and L-selectin knockout T cells but was significantly higher on L-selectin expressing T cells, particularly in the spleen. Clonal expansion of isolated L-selectin enhanced T cells was slower, and L-selectin was linked to expression of proliferation marker Ki67. Together these findings demonstrate that maintaining L-selectin expression on tumor-specific T cells offers an advantage in mouse models of cancer immunotherapy. The beneficial role of L-selectin is unrelated to its' well-known role in T cell homing and, instead, linked to activation of therapeutic T cells inside tumors. These findings suggest that L-selectin may benefit clinical applications in T cell selection for cancer therapy and for modifying CAR-T cells to broaden their clinical scope.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.