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Supplementary Data from Suppression of Endothelial Cell FAK Expression Reduces Pancreatic Ductal Adenocarcinoma Metastasis after Gemcitabine Treatment

Supplementary legends

Microarray data analysis

PML and HIF1a expression

Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA-damaging therapy–induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing, or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared with gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated patients with PDAC, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome.Significance:These findings establish the potential utility of combinatorial endothelial cell FAK targeting together with gemcitabine in future clinical applications to control metastasis in patients with pancreatic ductal adenocarcinoma.

New neuroblastoma patient cohort

Purpose: Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system with a complex biology, prone to metastasize and relapse. High-risk, metastatic cases are explained in part by amplification or mutation of oncogenes, such as MYCN and ALK, and loss of tumor suppressor genes in chromosome band 1p. However, it is fundamental to identify other pathways responsible for the large portion of neuroblastomas with no obvious molecular alterations.Experimental Design: Neuroblastoma cell lines were used for the assessment of tumor growth in vivo and in vitro. Protein expression in tissues and cells was assessed using immunofluorescence and IHC. The association of promyelocytic leukemia (PML) expression with neuroblastoma outcome and relapse was calculated using log-rank and Mann–Whitney tests, respectively. Gene expression was assessed using chip microarrays.Results: PML is detected in the developing and adult sympathetic nervous system, whereas it is not expressed or is low in metastatic neuroblastoma tumors. Reduced PML expression in patients with low-risk cancers, that is, localized and negative for the MYCN proto-oncogene, is strongly associated with tumor recurrence. PML-I, but not PML-IV, isoform suppresses angiogenesis via upregulation of thrombospondin-2 (TSP2), a key inhibitor of angiogenesis. Finally, PML-I and TSP2 expression inversely correlates with tumor angiogenesis and recurrence in localized neuroblastomas.Conclusions: Our work reveals a novel PML-I–TSP2 axis for the regulation of angiogenesis and cancer relapse, which could be used to identify patients with low-risk, localized tumors that might benefit from chemotherapy. Clin Cancer Res; 22(13); 3398–409. ©2016 AACR.

Pathway analysis

Supplementary Figure from Suppression of Endothelial Cell FAK Expression Reduces Pancreatic Ductal Adenocarcinoma Metastasis after Gemcitabine Treatment

Supplementary Figures S1-S7. Western blot quantification of pERK and pSTAT3 from aortic lysates (S1); Western blot quantification of pERK and pSTAT3 from MLEC lysates (S2); Western blot analysis and quantification of pERK and pSTAT3 from HUVEC lysates (S3); IL-6 reduces the expression of NG2 in the aortic ring vessels (S4); Regulation of Notch signaling and Angiopoietin2 by VEGF and IL-6 (S5); Effects of VEGF and IL-6 treatment on HUVEC (S6); Effects of anti-IL-6 antibody on in vivo IGROV-1 vasculature (S7).

Legend for Supplementary Figures S1-S7.

Evidence has emerged that bone marrow cells have a greater degree of plasticity than previously thought. However, there has been a call to establish proof that these bone marrow–derived cells function appropriately in their new environment. We have already shown that the bone marrow contributes to myofibroblasts in multiple organs and that this is exacerbated by injury and occurs in a mouse tumor model. Here, we provide evidence that these cells are functioning appropriately by showing that bone marrow–derived myofibroblasts are expressing mRNA for the α1 chain of type I (pro)collagen using a new customized technique. This provides evidence that the bone marrow-tumor stroma axis is functionally relevant and may therefore subsequently be exploited to develop new strategies for anticancer therapy. (Cancer Res 2006; 66(3): 1265-9)

Supplementary Figure 1 from Bone Marrow–Derived Stromal Cells Express Lineage-Related Messenger RNA Species

Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA-damaging therapy–induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing, or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared with gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated patients with PDAC, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome. Significance: These findings establish the potential utility of combinatorial endothelial cell FAK targeting together with gemcitabine in future clinical applications to control metastasis in patients with pancreatic ductal adenocarcinoma.

A correction to this paper has been published: https://doi.org/10.1007/s10456-021-09802-9

Background and aims The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer. Methods Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth. Results Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation. Conclusions This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology., Graphical abstract

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancer types and in vivo studies have shown that vascular endothelial cell FAK expression and FAK-phosphorylation at tyrosine (Y) 397, and subsequently FAK-Y861, are important in tumour angiogenesis. Pericytes also play a vital role in regulating tumour blood vessel stabilisation, but the involvement of pericyte FAK-Y397 and FAK-Y861 phosphorylation in tumour blood vessels is unknown. Using PdgfrβCre+;FAKWT/WT, PdgfrβCre+;FAKY397F/Y397F and PdgfrβCre+;FAKY861F/Y861F mice, our data demonstrate that tumour growth, tumour blood vessel density, blood vessel perfusion and pericyte coverage were affected only in late stage tumours in PdgfrβCre+;FAKY861F/Y861F but not PdgfrβCre+;FAKY397F/Y397F mice. Further examination indicates a dual role for pericyte FAK-Y861 phosphorylation in the regulation of tumour vessel regression and also in the control of a pericyte derived ‘pericrine’ signals that influence apoptosis in cancer cells. Overall this study identifies the role of pericyte FAK-Y861 in the regulation of tumour vessel regression and tumour growth control and that non-phosphorylatable FAK-Y861F in pericytes reduces tumour growth and blood vessel density.

The angiopoietin (Angpt)–TIE signaling pathway controls vascular maturation and maintains the quiescent phenotype of resting vasculature. The contextual agonistic and antagonistic Tie2 ligand ANGPT2 is believed to be exclusively produced by endothelial cells, disrupting constitutive ANGPT1–TIE2 signaling to destabilize the microvasculature during pathologic disorders like inflammation and cancer. However, scattered reports have also portrayed tumor cells as a source of ANGPT2. Employing ISH-based detection of ANGPT2, we found strong tumor cell expression of ANGPT2 in a subset of patients with melanoma. Comparative analysis of biopsies revealed a higher fraction of ANGPT2-expressing tumor cells in metastatic versus primary sites. Tumor cell–expressed Angpt2 was dispensable for primary tumor growth, yet in-depth analysis of primary tumors revealed enhanced intratumoral necrosis upon silencing of tumor cell Angpt2 expression in the absence of significant immune and vascular alterations. Global transcriptional profiling of Angpt2-deficient tumor cells identified perturbations in redox homeostasis and an increased response to cellular oxidative stress. Ultrastructural analyses illustrated a significant increase of dysfunctional mitochondria in Angpt2-silenced tumor cells, thereby resulting in enhanced reactive oxygen species (ROS) production and downstream MAPK stress signaling. Functionally, enhanced ROS in Angpt2-silenced tumor cells reduced colonization potential in vitro and in vivo. Taken together, these findings uncover the hitherto unappreciated role of tumor cell–expressed ANGPT2 as an autocrine-positive regulator of metastatic colonization and validate ANGPT2 as a therapeutic target for a well-defined subset of patients with melanoma. Significance: This study reveals that tumor cells can be a source of ANGPT2 in the tumor microenvironment and that tumor cell-derived ANGPT2 augments metastatic colonization by protecting tumor cells from oxidative stress.

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John WileySons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Simple Summary Although immunotherapy has given the highest rate of improvement in cancer treatment in recent years, there is an urgent need to further improve its efficacy. Numerous strategies aim to transform non-responsive, immunosuppressive tumours into sensitive, immunopermissive tumours. The modulation of the tumour microenvironment, and especially the tumour vasculature offers opportunities for improved sensitivity to immunotherapy. Modulation of tumour blood vessels can enhance tumour oxygenation and T cell infiltration. Additionally, maturation of tumour blood vessels is thought to be involved in the efficient delivery of therapeutic agents. This review compiles the current strategies of vascular modulation to improve the efficacy of different immunotherapies: PD-1/PD-L1 and CTLA-4 antibodies, CAR T cells and cancer vaccines. Abstract Several strategies have been developed to modulate the tumour vasculature for cancer therapy including anti-angiogenesis and vascular normalisation. Vasculature modulation results in changes to the tumour microenvironment including oxygenation and immune cell infiltration, therefore lending itself to combination with cancer therapy. The development of immunotherapies has led to significant improvements in cancer treatment. Particularly promising are immune checkpoint blockade and CAR T cell therapies, which use antibodies against negative regulators of T cell activation and T cells reprogrammed to better target tumour antigens, respectively. However, while immunotherapy is successful in some patients, including those with advanced or metastatic cancers, only a subset of patients respond. Therefore, better predictors of patient response and methods to overcome resistance warrant investigation. Poor, or periphery-limited, T cell infiltration in the tumour is associated with poor responses to immunotherapy. Given that (1) lymphocyte recruitment requires leucocyte–endothelial cell adhesion and (2) the vasculature controls tumour oxygenation and plays a pivotal role in T cell infiltration and activation, vessel targeting strategies including anti-angiogenesis and vascular normalisation in combination with immunotherapy are providing possible new strategies to enhance therapy. Here, we review the progress of vessel modulation in enhancing immunotherapy efficacy.

Coronary microvascular dysfunction combined with maladaptive cardiomyocyte morphology and energetics is a major contributor to heart failure advancement. Thus, dually enhancing cardiac angiogenesis and targeting cardiomyocyte function to slow, or reverse, the development of heart failure is a logical step towards improved therapy. We present evidence for the potential to repurpose a former anti‐cancer Arg‐Gly‐Asp (RGD)‐mimetic pentapeptide, cilengitide, here used at low doses. Cilengitide targets αvβ3 integrin and this protein is upregulated in human dilated and ischaemic cardiomyopathies. Treatment of mice after abdominal aortic constriction (AAC) surgery with low‐dose cilengitide (ldCil) enhances coronary angiogenesis and directly affects cardiomyocyte hypertrophy with an associated reduction in disease severity. At a molecular level, ldCil treatment has a direct effect on cardiac endothelial cell transcriptomic profiles, with a significant enhancement of pro‐angiogenic signalling pathways, corroborating the enhanced angiogenic phenotype after ldCil treatment. Moreover, ldCil treatment of Angiotensin II‐stimulated AngII‐stimulated cardiomyocytes significantly restores transcriptomic profiles similar to those found in normal human heart. The significance of this finding is enhanced by transcriptional similarities between AngII‐treated cardiomyocytes and failing human hearts. Taken together, our data provide evidence supporting a possible new strategy for improved heart failure treatment using low‐dose RGD‐mimetics with relevance to human disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCre+;FAKY397F/Y397F–mutant mice. Conversely, ECCre+;FAKY861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased β1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in β1 integrin activation. This corresponds with a decrease in Vegfa–stimulated response, but an increase in Vegfa+Ang2- or conditioned medium from tumor cell–stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to Vegfa+Ang2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. Significance: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

Key Points Question Are phosphorylated–focal adhesion kinase (pY397-FAK) expression levels in endothelial or tumor cells and tumor blood vessel density associated with response to chemotherapy and relapse-free survival after neoadjuvant chemotherapy in patients with locally advanced breast cancer? Findings In this prognostic study of 82 women with stage IIA to IIIC locally advanced breast cancer, low endothelial cell pY397-FAK expression levels in prechemotherapy core biopsies were associated with sensitivity to neoadjuvant chemotherapy and improved 5-year relapse-free survival. Combined analysis of high endothelial cell pY397-FAK, high tumor cell pY397-FAK, and high blood vessel density appeared to identify a high-risk population. Meaning The findings of this study suggest that endothelial cell pY397-FAK levels could be used as a clinical tool for indicating response to neoadjuvant chemotherapy., Importance Determining the risk of relapse after neoadjuvant chemotherapy in patients with locally advanced breast cancer is required to offer alternative therapeutic strategies. Objective To examine whether endothelial cell phosphorylated–focal adhesion kinase (EC-pY397-FAK) expression in patients with treatment-naive locally advanced breast cancer is a biomarker for chemotherapy sensitivity and is associated with survival after neoadjuvant chemotherapy. Design, Setting, and Participants In this prognostic study, expression levels of EC-pY397-FAK and tumor cell (TC)-pY397-FAK were determined by immunohistochemistry in prechemotherapy core biopsies from 82 female patients with locally advanced breast cancer treated with anthracycline-based combination neoadjuvant chemotherapy at Nottingham City Hospital in Nottingham, UK. Median follow-up time was 67 months. The study was conducted from December 1, 2010, to September 28, 2019, and data analysis was performed from October 2, 2019, to March 31, 2020. Exposures All women underwent surgery followed by adjuvant radiotherapy and, if tumors were estrogen receptor–positive, 5-year tamoxifen treatment. Main Outcomes and Measures Outcomes were pathologic complete response and 5-year relapse-free survival examined using Kaplan-Meier, univariable logistic, multivariable logistic, and Cox proportional hazards models. Results A total of 82 women (age, 29-76 years) with locally advanced breast cancer (stage IIA-IIIC) were included. Of these, 21 women (26%) had high EC-pY397-FAK expression that was associated with estrogen receptor positivity (71% vs 46%; P = .04), progesterone receptor positivity (67% vs 39%; P = .03), high Ki67 (86% vs 41%; P, This prognostic study examines the use of endothelial cell phosphorylated-focal adhesion kinase as a biomarker for chemotherapy sensitivity in women with breast cancer.

Ischaemic heart disease remains a leading global cause of death. Restoring blood perfusion in the peri-infarct border region may limit further infarct expansion and promote cardiac regeneration. However, the pathways driving endogenous vascular regeneration following myocardial infarction (MI) remain poorly understood. We aimed to investigate the origin, clonal dynamics and transcriptional profiles of resident coronary endothelial cells (EC) in the post-ischaemic adult mouse heart and to apply single cell RNA-sequencing (scRNAseq) to identify and validate novel targets with a potential role in neovasculogenesis following MI. MI was induced in an EC-specific multispectral lineage-tracing mouse, ‘Pdgfb-iCreERT2-R26R-Brainbow2.1’ by permanent ligation of the left anterior descending coronary artery. Blood vessel formation via clonal proliferation by resident coronary vascular EC was significantly upregulated in the ischaemic border at 7 days post-MI, compared to the healthy heart (Pdgfb-Confetti+ EC per clone = 4.0 ± 2.1 versus 10.3 ± 10.6, P

Emerging evidence suggests that cancer cell metabolism can be regulated by cancer-associated fibroblasts (CAFs), but the mechanisms are poorly defined. Here we show that CAFs regulate malignant cell metabolism through pathways under the control of FAK. In breast and pancreatic cancer patients we find that low FAK expression, specifically in the stromal compartment, predicts reduced overall survival. In mice, depletion of FAK in a subpopulation of CAFs regulates paracrine signals that increase malignant cell glycolysis and tumour growth. Proteomic and phosphoproteomic analysis in our mouse model identifies metabolic alterations which are reflected at the transcriptomic level in patients with low stromal FAK. Mechanistically we demonstrate that FAK-depletion in CAFs increases chemokine production, which via CCR1/CCR2 on cancer cells, activate protein kinase A, leading to enhanced malignant cell glycolysis. Our data uncover mechanisms whereby stromal fibroblasts regulate cancer cell metabolism independent of genetic mutations in cancer cells., Cancer associated fibroblasts (CAFs) have been suggested to regulate cancer cell metabolism, but the mechanisms are not completely elucidated. Here, the authors show that low FAK expression in stromal cells correlates with poor prognosis in breast and pancreatic cancer patients and that FAK-silencing in CAFs promotes tumourigenesis by the paracrine regulation of cancer cell metabolism.

During obesity, macrophages infiltrate the breast tissue leading to low‐grade chronic inflammation, a factor considered responsible for the higher risk of breast cancer associated with obesity. Here, we formally demonstrate that breast epithelial cells acquire malignant properties when exposed to medium conditioned by macrophages derived from human healthy donors. These effects were mediated by the breast cancer oncogene IKKε and its downstream target—the serine biosynthesis pathway as demonstrated by genetic or pharmacological tools. Furthermore, amlexanox, an FDA‐approved drug targeting IKKε and its homologue TBK1, delayed in vivo tumour formation in a combined genetic mouse model of breast cancer and high‐fat diet‐induced obesity/inflammation. Finally, in human breast cancer tissues, we validated the link between inflammation–IKKε and alteration of cellular metabolism. Altogether, we identified a pathway connecting obesity‐driven inflammation to breast cancer and a potential therapeutic strategy to reduce the risk of breast cancer associated with obesity., In this article a new pathway has been identified that links macrophage‐driven inflammation and cellular metabolism to the acquisition of malignant traits in breast epithelium providing new insight into the well‐established association between breast cancer and obesity and offering a new preventive strategy.

Pathological angiogenesis contributes to tumour progression as well as to chronic inflammatory diseases. In this issue of EMBO Molecular Medicine, Esteban and co‐workers identify endothelial cell MT1‐MMP as a key regulator of intussusceptive angiogenesis (IA) in inflammatory colitis. Thrombospondin 1 (TSP1) cleavage by MT1‐MMP results in the binding of the c‐terminal fragment of TSP1 to αvβ3 integrin, which induces nitric oxide (NO) production, vasodilation and further initiation of IA. This novel control mechanism of inflammatory IA points towards promising new therapeutic targets for inflammatory bowel disease., Prof. Hodivala‐Dilke and colleagues discuss the findings from Esteban et al (in this issue of EMBO Molecular Medicine) that targeting intussusceptive angiogenesis via the TSP1‐nitric oxide axis exhibits therapeutic efficacy in the context of inflammatory bowel disease.

Aims: A better understanding of the pathways that regulate regeneration of the coronary vasculature is important for future strategies to treat patients with heart disease. We investigated (i) the clonal dynamics of endothelial cells (EC) associated with neovascularization in the ischemic border region (ii) transcriptional signatures of regenerative EC in the ischemic heart using single cell RNA-sequencing (iii) the functional relevance of selected targets. Methods: MI was induced in ‘EC-Confetti’ mice by coronary artery ligation. EC clonal proliferation was quantified or hearts dissociated for scRNAseq. Immunofluorescence staining for targets identified by scRNAseq was performed on cardiac tissue from patients with ischemic heart disease. EC proliferation was assessed in vitro following siRNA gene silencing. Results: EC-Confetti mice express YFP, RFP, GFP, or CFP specifically in EC. Fluorophores are inherited by EC progeny following proliferation, allowing quantitative clonal analysis. Clonal proliferation was significantly increased in the infarct border at 7 days post-MI compared to healthy hearts ( P P =0.002) and mouse ( P =0.002) hearts, compared to healthy myocardium. siRNA gene silencing of Plvap significantly inhibited EC proliferation ( P = 0.0038), strong evidence that Plvap can directly modulate EC function. Conclusions: Generation of new blood vessels following ischemic injury in the mouse heart is predominantly mediated by clonal proliferation of resident EC. We present a gene expression atlas of resident cardiac EC, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. This resource identifies novel targets, including Plvap, that may augment myocardial perfusion post-MI, and inform future design of strategies aimed at promoting vascular perfusion in ischemic heart disease.

[Image: see text] The integrin αVβ3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC(50), which correlates with cilengitide’s enhancement of tumor growth in vivo.

Aims C-type natriuretic peptide (CNP) is an essential endothelium-derived signalling species that governs vascular homoeostasis; CNP is also expressed in the heart but an intrinsic role for the peptide in cardiac function is not established. Herein, we employ unique transgenic strains with cell-specific deletion of CNP to define a central (patho)physiological capacity of CNP in maintaining heart morphology and contractility. Methods and results Cardiac structure and function were explored in wild type (WT), cardiomyocyte (cmCNP−/−), endothelium (ecCNP−/−), and fibroblast (fbCNP−/−)—specific CNP knockout mice, and global natriuretic peptide receptor (NPR)-B−/−, and NPR-C−/− animals at baseline and in experimental models of myocardial infarction and heart failure (HF). Endothelium-specific deletion of CNP resulted in impaired coronary responsiveness to endothelium-dependent- and flow-mediated-dilatation; changes mirrored in NPR-C−/− mice. Ex vivo, global ischaemia resulted in larger infarcts and diminished functional recovery in cmCNP−/− and NPR-C−/−, but not ecCNP−/−, vs. WT. The cardiac phenotype of cmCNP−/−, fbCNP−/−, and NPR-C−/− (but not ecCNP−/− or NPR-B−/−) mice was more severe in pressure overload- and sympathetic hyperactivation-induced HF compared with WT; these adverse effects were rescued by pharmacological CNP administration in WT, but not NPR-C−/−, mice. At a molecular level, CNP/NPR-C signalling is impaired in human HF but attenuates activation of well-validated pro-hypertrophic and pro-fibrotic pathways. Conclusion C-type natriuretic peptide of cardiomyocyte, endothelial and fibroblast origins co-ordinates and preserves cardiac structure, function, and coronary vasoreactivity via activation of NPR-C. Targeting NPR-C may prove an innovative approach to treating HF and ischaemic cardiovascular disorders.

The nonreceptor protein tyrosine kinase, focal adhesion kinase (FAK, also known as PTK2), is a key mediator of signal transduction downstream of integrins and growth factor receptors in a variety of cells, including endothelial cells. FAK is upregulated in several advanced-stage solid tumors and has been described to promote tumor progression and metastasis through effects on both tumor cells and stromal cells. This observation has led to the development of several FAK inhibitors, some of which have entered clinical trials (GSK2256098, VS-4718, VS-6062, VS-6063, and BI853520). Resistance to chemotherapy is a serious limitation of cancer treatment and, until recently, most studies were restricted to tumor cells, excluding the possible roles performed by the tumor microenvironment. A recent report identified endothelial cell FAK (EC-FAK) as a major regulator of chemosensitivity. By dysregulating endothelial cell–derived paracrine (also known as angiocrine) signals, loss of FAK solely in the endothelial cell compartment is able to induce chemosensitization to DNA-damaging therapies in the malignant cell compartment and thereby reduce tumor growth. Herein, we summarize the roles of EC-FAK in cancer and development and review the status of FAK-targeting anticancer strategies. Clin Cancer Res; 22(15); 3718–24. ©2016 AACR.

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-β3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-β3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell β3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by β3-integrin, providing a previously unrecognized mechanism of cancer growth control.

Despite the onset of new treatment strategies, mortality rates for breast cancer are estimated at approximately 600,000 women per year, mostly due to acquired resistance to therapy. Chemotherapy, often combination therapy, includes DNA-damaging agents such as epirubicin, an epimer of doxorubicin. Although chemotherapy has been reported to reduce recurrence rates by 50% over two years, eight-year survival rates are approximately 30% and overall mortality is 20-25%. Studies aiming to overcome resistance to therapy have focused mainly on in vitro malignant cell assays. These have failed to deliver appropriate targets to overcome resistance because they have failed to account for the influence of the tumor microenvironment on chemoresistance. Published evidence indicates that resistance to therapy is not malignant cell-intrinsic, but instead depends on DNA-damaging chemotherapy-induced changes in paracrine signals from endothelial cells. Our current research has established that the doxorubicin treatment acutely affects endothelial cells with long-term effects on tumor cell resistance to therapy in vivo. Endothelial cells are heterogenous, and the effect of DNA damage on endothelial cell evolution and how this regulates malignant cell resistance to therapy is unknown. We have observed that orthotopic murine mammary tumors arising from the same source of primary mammary tumor cells show differential chemotherapy responses. RNA seq analysis of tumors after acute doxorubicin treatment reveal highest levels of differential transcript expression in endothelial cells compared with cancer-associated fibroblasts or malignant cells. In vitro assays show that conditioned media from tumor endothelial cells treated with chemotherapy can protect tumor cells from DNA damage and cell death. Current analysis now focuses on analyzing the complexity of endothelial cell regulation of tumor cell response to chemotherapy and how this could model clonal evolution of tumor cells. We aim to create a chemoresistance stromal evolution signature, to determine the individual components of this and commonalities between cancers and possible drug effects. Thus, our data could change the paradigm of drug resistance and define how stromal compartments regulate acquired resistance. Citation Format: Jesus Gomez-Escudero, Eleni Maniati, Freddie Whiting, Jun Wang, Trevor Graham, Kairbaan Hodivala-Dilke. Tumor stroma in the development of acquired cancer therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A31.

The cytokine IL6 has a number of tumor-promoting activities in human and experimental cancers, but its potential as an angiogenic agent has not been fully investigated. Here, we show that IL6 can directly induce vessel sprouting in the ex vivo aortic ring model, as well as endothelial cell proliferation and migration, with similar potency to VEGF. However, IL6-stimulated aortic ring vessel sprouts had defective pericyte coverage compared with VEGF-stimulated vessels. The mechanism of IL6 action on pericytes involved stimulation of the Notch ligand Jagged1 as well as angiopoietin2 (Ang2). When peritoneal xenografts of ovarian cancer were treated with an anti-IL6 antibody, pericyte coverage of vessels was restored. In addition, in human ovarian cancer biopsies, there was an association between levels of IL6 mRNA, Jagged1, and Ang2. Our findings have implications for the use of cancer therapies that target VEGF or IL6 and for understanding abnormal angiogenesis in cancers, chronic inflammatory disease, and stroke. Cancer Res; 75(15); 3098–107. ©2015 AACR.

We describe a systematic approach for the development of both orally bioavailable and bioactive cyclic N methylated hexapeptides as high affinity ligands for the integrin αvβ3. The work is based on two concepts: (a) screening of systematically designed libraries with spatial diversity and (b) masking of the peptide charge with lipophilic pro-moiety. The key steps of the method are i) initial design of combinatorial library of N-methylated analogs of the stem peptide cyclo(D Ala Ala5); ii) selection of cyclic peptides with highest intestinal permeability; iii) design of sub-libraries with the bioactive RGD sequence in all possible positions; iv) selection of the best ligands for RGD-recognizing integrin subtypes; v) fine tuning of affinity and selectivity by additional Ala to Xaa substitutions; vi) protection of the charged functional groups according to the pro drug concept to regain intestinal and oral permeability; vii) proof of biological effects in mice after oral administration

Summary Focal adhesion kinase is a non-receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non-kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK-mutant knockin mice where mutant FAK, either kinase dead, non-phosphorylatable mutants Y397F and Y861F, or mutant Y397E—containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre-expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism. genesis 52:907–915, 2014. © 2014 Wiley Periodicals, Inc.

Rationale: The dramatic upregulation of αvβ3-integrin that occurs in the vasculature during tumor growth has long suggested that the endothelial expression of this molecule is an ideal target for antiangiogenic therapy to treat cancer. This discovery led to the development of small-molecule inhibitors directed against αvβ3-integrin that are currently in clinical trials. In 2002, we reported that β3-integrin−knockout mice exhibit enhanced tumor growth and angiogenesis. However, as β3-integrin is expressed by a wide variety of cells, endothelial cell–specific contributions to tumor angiogenesis are muddied by the use of a global knockout of β3-integrin function. Objective: Our aim was to examine the endothelial-specific contribution β3-integrin makes to tumor growth and angiogenesis. Methods and Results: We have crossed β3-integrin–floxed (β3-floxed) mice to 2 endothelial-specific Cre models and examined angiogenic responses in vivo, ex vivo, and in vitro. We show that acute depletion of endothelial β3-integrin inhibits tumor growth and angiogenesis preventatively, but not in already established tumors. However, the effects are transient, and long-term depletion of the molecule is ineffective. Furthermore, long-term depletion of the molecule correlates with many molecular changes, such as reduced levels of focal adhesion kinase expression and a misbalance in focal adhesion kinase phosphorylation, which may lead to a release from the inhibitory effects of decreased endothelial β3-integrin expression. Conclusions: Our findings imply that timing and length of inhibition are critical factors that need to be considered when targeting the endothelial expression of β3-integrin to inhibit tumor growth and angiogenesis.

The extracellular protease ADAMTS1 (A disintegrin and metalloprotease with thrombospondin repeats 1) has been described as an anti-angiogenic molecule and its role as a putative tumor protective molecule has also been suggested. Here, we have used a tumor xenograft model to determine the role of ADAMTS1 in tumor growth and angiogenesis. Increasing levels of the protease led to the complete inhibition of tumor growth. In an attempt to elucidate the mechanism of action of this protease, we focused our attention on its proteolytic activity on nidogens, one of the main components of the vascular basement membrane. The increased expression of ADAMTS1 was accompanied by increased proteolysis of nidogen-1 and -2 and their almost complete removal from vascular structures, together with major morphological alterations of tumor blood vessels and a decreased vessel density. The clinical relevance of this work is supported by our observations that ADAMTS1 expression is decreased in breast tumor specimens when compared with healthy tissue. Our studies also reveal that the cleavage of nidogen-1 and -2 is partially inhibited in human tumor samples. Moreover, the deposition of both nidogens surrounding vascular structures is drastically altered, implying a possible reduction in the maintenance of vessel integrity. Our studies reflect the requirement to explore the functional interactions between proteases and specific substrates in cancer biology.