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Background The disappointing activity of single agent immune-checkpoint inhibitors in epitherlial ovarian cancer (EOC) has been attributed in part to its unique tumor microenvironment (TME). IDO, PDL1, LAG3 and TIM3 have been implicated in the immunotolerance of EOC. We investigated the expression of these co-regulators, their change with neoadjuvant chemotherapy (NACT), and their association with outcome. Method We identified 98 patients with EOC treated with NACT and performed IDO, PDL1, LAG3 and TIM3 immunohistochemistry on samples obtained before and after NACT. The cut-off threshold to consider a positive sample was set at 5%. Results In our cohort, TIM3 was the most prevalent co-regulator, with more than 75% of the samples being TIM3 positive. In comparison, only 22%, 28% and 17% of the samples were considered IDO, PDL1 and LAG3 positive. More than half of ovarian tumors expressed 2, 3 or even all 4 co-inhibitory molecules. However, biomarkers were not correlated with each other. NACT had a marked impact on immune co-regulator expression with over 70% of patients showing a change in biomarker status from negative to positive or vice versa. There was no significant difference in the pattern of co-regulator expression between platinum-sensitive and resistant patients. Co-expression of multiple inhibitory molecules did not appear to affect overall and progression-free survival. Conclusion TIM3 is the most abundant co-inhibitory molecule in OC and may represent an attractive target. In addition, OC frequently co-expressed 2 or more markers supporting ICI combinatorial approaches. Finally, NACT significantly altered the expression of immunosuppressive molecules suggesting that the choice of ICI combinations should be adapted to the composition of the post-NACT immune TME.

5554 Background: Antibodies targeting PDL1 or PD1 have been disappointing so far in the treatment of ovarian cancer (OC). A greater understanding of the complex iTME and of the impact of chemotherapy on immune features could uncover promising immune targets. We previously reported that neoadjuvant chemotherapy (NACT) increased CD4+ and CD8+ immune cells (IC) and depleted FOXP3+ suppressive T-regs in OC iTME. Here we aimed to describe the expression of PDL1 as well as other co-regulatory molecules in OC and their changes under NACT. Methods: Tumor samples and clinical data were prospectively collected from patients (pts) in the randomized CHIVA trial of NACT +/- nintedanib. Samples were evaluable for immune profiling for 116-124 pts at diagnosis and 89-107 at surgery after 3 cycles of NACT. IC stained for CD4, CD8 were scored as number of IC+/mm². Expression of immune co-regulatory molecules PDL1, TIM3, LAG3 and IDO was scored as percentage of positive cells, and tumors were classified as PDL1/TIM3/IDO/LAG3 positive if > 1% of IC and/or tumor cells (TC) were positive. Highly sensitive pts, defined as objective response to NACT and prolonged median progression-free survival (mPFS > 24months), were compared to refractory pts (progressing during or within 3mo of platinum). Results: As expected, about one third (36%) of tumors were PDL1+ at diagnosis. In contrast, the prevalence of other co-regulatory molecules was higher with 52%, 54% and 93% of tumors being positive for IDO, LAG3 and TIM3, respectively. There was no significant change in PDL1 expression with NACT. However, in paired samples NACT significantly increased IDO and LAG3 expression (p < 0.05), such that 60% and 66% of tumors post-NACT were positive for IDO and LAG3, respectively. TIM3 expression remained high post-NACT with 92% of positive tumors. Highly sensitive tumors (vs refractory tumors) had significantly higher IC expression of TIM3 after NACT (24% vs 6%, p = 0.005), and were significantly more infiltrated by CD4+ (441 vs 228 cells/mm2, p = 0.04) and CD8+ (460 vs 225 cells/mm2, p = 0.045) T cells. Conclusions: Other immune targets beyond PDL1 are highly expressed in OC. In addition NACT appears to prime the iTME by increasing effector T cell infiltration and the expression of other relevant co-regulatory molecules (LAG3, TIM3 and IDO). Future studies could be performed by priming the iTME with NACT and testing novel immune therapies based on target expression in samples obtained at interval debulking surgery.

Background Activated cardiac fibroblasts produce extracellular matrix proteins that accumulate during cardiac fibrosis. We have recently shown that PW1 is expressed in a subset of cardiac stromal cells and that cardiac PW1+ cells represent a cellular source of fibroblasts in the ischemic hearts. Purpose We aimed to further identify new cell surface markers expressed by cardiac PW1+ cells and to investigate their role in the fibrogenic behavior of these cells. Methods and results We first performed transcriptomic and proteomic profiling of FACS-isolated cardiac PW1+ from normal and ischemic hearts. RNA-sequencing output files were processed with bioinformatics algorithms to identify 378 specific cell-surface markers for cardiac PW1+ cells. By comparing these candidates with the proteomic profile, we then cross-identified 9 cell surface proteins primarily involved in cell motility, adhesion to the matrix, inflammatory response and response to wounding. One of these candidates (i.e., aV-integrin or CD51) was expressed in almost all cardiac PW1+ cells (93±1%), and was predominantly found in cells expressing PW1 in the myocardium. Cardiac PW1+ cells showed a predominant expression of aVβ1 complex which is known to mediate fibrosis through TGF-beta activation in a number of tissues. The transfer of isolated cardiac PW1+CD51+ cells into ischemic hearts was associated with fibrosis development. We further demonstrated that inhibition of aV-integrin in cardiac PW1+ cells reduces their profibrotic gene expression profile and their ability to differentiate into fibroblasts. Lastly, a pharmacological blockade of aV-integrin improved cardiac function and animal survival following myocardial infarction coupled with a reduced infarct size and fibrotic lesion. Conclusions These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of aV-integrin leads to reduced pathological outcomes following cardiac ischemia. Acknowledgement/Funding Fondation Leducq (grant 13CVD01, CardioStemNet project), Fédération Française de Cardiologie and Era-CVD (ANR-16-ECVD-0011-03, Clarify project)

Rationale: Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1 + progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro. Objective: To determine the role of pulmonary PW1 + progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension. Methods and Results: We investigated their contribution during chronic hypoxia–induced vascular remodeling in Pw1 nLacZ+/− mouse expressing β-galactosidase in PW1 + cells and in differentiated cells derived from PW1 + cells. PW1 + progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1 + cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1 + cells and the proportion of β-gal + vascular SMC were increased, indicating a recruitment of PW1 + cells and their differentiation into vascular SMC during early chronic hypoxia–induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1 + cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed β-gal + SMC were not derived from circulating bone marrow–derived PW1 + progenitor cells, confirming a resident origin of the recruited PW1 + cells. The number of pulmonary PW1 + cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures. Conclusions: These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension–associated vascular remodeling.

Background—Stem cell factor (SCF), a ligand of the c-kit receptor, is a critical cytokine, which contributes to cell migration, proliferation, and survival. It has been shown that SCF expression increases after myocardial infarction (MI) and may be involved in cardiac repair. The aim of this study was to determine whether gene transfer of membrane-bound human SCF improves cardiac function in a large animal model of MI. Methods and Results—A transmural MI was created by implanting an embolic coil in the left anterior descending artery in Yorkshire pigs. One week after the MI, the pigs received direct intramyocardial injections of either a recombinant adenovirus encoding for SCF (Ad.SCF, n=9) or β-gal (Ad.β-gal, n=6) into the infarct border area. At 3 months post-MI, ejection fraction increased by 12% relative to baseline after Ad.SCF therapy, whereas it decreased by 4.2% (P=0.004) in pigs treated with Ad.β-gal. Preload-recruitable stroke work was significantly higher in pigs after SCF treatment (Ad.SCF, 55.5±11.6 mm Hg versus Ad.β-gal, 31.6±12.6 mm Hg, P=0.005), indicating enhanced cardiac function. Histological analyses confirmed the recruitment of c-kit[superscript +] cells as well as a reduced degree of apoptosis 1 week after Ad.SCF injection. In addition, increased capillary density compared with pigs treated with Ad.β-gal was found at 3 months and suggests an angiogenic role of SCF. Conclusions—Local overexpression of SCF post-MI induces the recruitment of c-kit[superscript +] cells at the infarct border area acutely. In the chronic stages, SCF gene transfer was associated with improved cardiac function in a preclinical model of ischemic cardiomyopathy., National Institutes of Health (U.S.) (R01 HL117505), National Heart, Lung, and Blood Institute (Program of Excellence in Nanotechnology (PEN) Award Contract HHSN268201000045C), P50 HL112324, Leducq Foundation

Rationale: Adult myocardium contains a rare population of cardiac progenitor cells (CPCs), recruited in response to tissue injury, which are of central importance for the cardiac microenvironment after cardiac injury. However, their phenotype characterization and function in physiological and pathological has not yet been reported. Objective: Based on the identification of a cell population with very high expression of PW1 in different tissues, our goal was to determine the role that PW1+ cells play in the heart in a murine myocardial infarction model. Methods and Results: In this study we show that high expression of PW1 in cardiac cells (PW1+ cells), identified a rare population, located in the epicardium and interstitium in resting heart, increased in the injured and border area in response to myocardial infarction (MI). Using PW1-reporter mice, we defined three populations of CPCs that include fraction I (PW1-ckit+), II (PW1+ c-kit+) and III (PW1+ckit-) based on differential expression of PW1 and c-kit. We found that MI led to an increase of all the fractions especially fraction II. Furthermore, isolated PW1+ cells showed clonogenic capacity, measured as fibroblastic colony-forming units frequency, and differentiation capacity to give rise in vitro to several cardiac lineages. The phenotypic characterization of fractions I, II and III has also been studied by utilizing comparative gene expression profile as a screening tool. Taken together, these data identify a novel PW1+ cardiac progenitor population with the potential to undergo differentiation into multiple cardiac lineages, suggesting their involvement in cardiac repair in normal and pathological conditions. Conclusion: Based on the comparative phenotype analysis, we could demonstrate that the expression of PW1 identified a pure population of CPCs, augmented following MI, with clonogenic and multipotent capacity, which is likely to provide a novel target for therapeutic approaches aimed at improving cardiac repair.

The aim of the present study is to explore the role of capillary disorder in coronary ischemic congestive heart failure (CHF). CHF was induced in rats by aortic banding plus ischemia-reperfusion followed by aortic debanding. Coronary arteries were perfused with plastic polymer containing fluorescent dye. Multiple fluorescent images of casted heart sections and scanning electric microscope of coronary vessels were obtained to characterize changes in the heart. Cardiac function was assessed by echocardiography and in vivo hemodynamics. Stenosis was found in all levels of the coronary arteries in CHF. Coronary vasculature volume and capillary density in remote myocardium were significantly increased in CHF compared with control. This occurred largely in microvessels with a diameter of ≤3 μm. Capillaries in CHF had a tortuous structure, while normal capillaries were linear. Capillaries in CHF had inconsistent diameters, with assortments of narrowed and bulged segments. Their surfaces appeared rough, potentially indicating endothelial dysfunction in CHF. Segments of main capillaries between bifurcations were significantly shorter in length in CHF than in control. Transiently increasing preload by injecting 50 μl of 30% NaCl demonstrated that the CHF heart had lower functional reserve; this may be associated with congestion in coronary microcirculation. Ischemic coronary vascular disorder is not limited to the main coronary arteries, as it occurs in arterioles and capillaries. Capillary disorder in CHF included stenosis, deformed structure, proliferation, and roughened surfaces. This disorder in the coronary artery architecture may contribute to the reduction in myocyte contractility in the setting of heart failure.

Understanding of the signal transduction pathways that lead to B cell development is of extreme interest to learn how alterations in these pathways might initiate malignant transformation. Long-term cultured early pre-BI cells can differentiate into IgM+ B cells after transplant into NOD/SCID mice, offering the possibility to explore checkpoints in B cell development. Using DNA microarray and Western blot analysis of IgM+ B cells vs parental early pre-BI cells, we demonstrated that zeta-associated protein 70 (ZAP-70) is upregulated in our B cell differentiation model. Unlike parental ZAP-70- early pre-BI cells, ZAP-70+ IgM+ B cells exhibited a transformed phenotype, as indicated by BCL-6 expression, a high Ki-67 proliferation index, resistance to IL-7 deprivation-induced apoptosis, and an increased repopulation rate in NOD/SCID mice. These data show the characterization and generation of a novel murine leukemia model with many similarities to human ZAP-70+ B cell chronic lymphocytic leukemia.

The myocardium responds to injury by recruiting cardiac progenitor cells (CPCs) to the injured tissue to promote cardiac repair. Although different classes of CPCs have been identified, their contribution in physiological and pathological conditions remains unclear. PW1 gene has recently been proposed as a marker of resident adult stem and progenitor cell populations in several adult tissues. Our goal was to characterize and determine the role of PW1+ population in the heart. Here, we employ immunostaining and fluorescence-activated cell sorting (FACS) analysis in PW1-reporter mouse to perform qualitative and quantitative analyses of PW1+ population in the heart. We first found that PW1+ cells are mainly located in the epicardium and myocardial interstitium of normal hearts. The average percentage of PW1+ cells, as assessed by FACS, was 1.56±1.41%. A subset of PW1+ cells also co-express other CPC markers such as Sca-1 (52±22%) or PDGFR1α (43±14%). In contrast, a very small proportion of PW1+ cells co-express c-kit (6±5%). To investigate the contribution of PW1+ cells in pathological conditions, we then performed myocardial infarction (MI) by LAD ligation in PW1-reporter mice. We found that MI resulted in a 3-fold increase in the number of PW1+ cells in infarcted mice compared with sham-operated groups, at 1 week post-MI (1.16%±0.47% in sham versus 3.43%±0.82 in MI). This population preferentially localized in the injured myocardium and border area. PW1+ cells were isolated by FACS from the whole infarcted heart from PW1-reporter mice. In vitro differentiation assays reveal that purified PW1+ cells are multipotent and can spontaneously differentiate into smooth muscle cells, endothelial cells and cardiomyocyte-like cells. Taken together, our data identify a novel PW1+ cardiac progenitor population with the potential to undergo differentiation into multiple cardiac lineages, suggesting their involvement in cardiac repair in normal and pathological conditions. The discovery of a novel population of cardiac progenitor cells, augmented following MI and with cardiogenic potential, provides a novel target for therapeutic approaches aimed at improving cardiac regeneration.

AIMS: Mammalian myocardium has a finite but limited capacity to regenerate. Experimentally stimulating proliferation of cardiomyocytes with extracellular regeneration factors like periostin enhances cardiac repair in rodents. The aim of this study was to develop a safe method for delivering regeneration factors to the heart and to test the functional and structural effects of periostin peptide treatment in a large animal model of myocardial infarction (MI). METHODS AND RESULTS: We developed a controlled release system to deliver recombinant periostin peptide into the pericardial space. A single application of this method was performed two days after experimental MI in swine. Animals were randomly assigned to receive either saline or periostin peptide. Experimental groups were compared at baseline, day 2, 1 month and 3 months. Treatment with periostin peptide increased the EF from 31% to 41% and decreased by 22% the infarct size within 12 weeks. Periostin peptide-treated animals had newly formed myocardium strips within the infarct scar, leading to locally improved myocardial function. In addition the capillary density was increased in animals receiving periostin. However, periostin peptide treatment increased myocardial fibrosis in the remote region at one week and 12 weeks post-treatment. CONCLUSION: Our study shows that myocardial regeneration through targeted peptides is possible. However, in the case of periostin the effects on cardiac fibrosis may limit its clinical application as a viable therapeutic strategy.

Rationale Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling and neomuscularization. PW1+ progenitor cells can differentiate into smooth muscle cells (SMC) in vitro. Objective To determine the role of pulmonary PW1+ progenitor cells in vascular remodeling characteristic of PAH. Methods and results We investigated their contribution during chronic hypoxia (CH)-induced vascular remodeling in Pw1nLacZ+/– mouse expressing β-galactosidase in PW1+ cells and in differentiated cells derived from PW1+ cells. PW1+ progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, three distinct myogenic PW1+ cell populations were isolated from the mouse lung of which two were significantly increased after 4 days of CH. The number of proliferating pulmonary PW1+ cells and the proportion of β-gal+ vascular SMC were increased, indicating a recruitment of PW1+ cells and their differentiation into vascular SMC during early CH-induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1+ cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed β-gal+ SMC were not derived from circulating bone marrow-derived PW1+ progenitor cells, confirming a resident origin of the recruited PW1+ cells. The number of pulmonary PW1+ cells was also increased in rats after monocrotaline (MCT) injection. In the human PAH lung, PW1-expressing cells were observed in large numbers in remodeled vascular structures. Conclusions These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in PAHassociated vascular remodeling. The author hereby declares no conflict of interest

This study investigates the impact of pressure overload on vascular changes after myocardial infarction (MI) in rats. To evaluate the effect of pressure overload, MI was induced in three groups: 1) left coronary artery ligation for 1 mo (MI-1m), 2) ischemia 30 min/reperfusion for 1 mo (I/R-1m), and 3) ischemia-reperfusion (I/R) was performed after pressure overload induced by aortic banding for 2 mo; 1 mo post-I/R, aortic constriction was released (Ab+I/R+DeAb). Heart function was assessed by echocardiography and in vivo hemodynamics. Resin casting and three-dimensional imaging with microcomputed tomography were used to characterize changes in coronary vasculature. TTC (triphenyltetrazohum chloride) staining and Masson's Trichrome were conducted in parallel experiments. In normal rats, MI induced by I/R and permanent occlusion was transmural or subendocardial. Occluded arterial branches vanished in MI-1m rats. A short residual tail was retained, distal to the occluded site in the ischemic area in I/R-1m hearts. Vascular pathological changes in transmural MI mostly occurred in ischemic areas and remote vasculature remained normal. In pressure overloaded rats, I/R injury induced a sub-MI in which ischemia was transmural, but myocardium in the involved area had survived. The ischemic arterial branches were preserved even though the capillaries were significantly diminished and the pathological changes were extended to remote areas, characterized by fibrosis, atrial thrombus, and pulmonary edema in the Ab+I/R+DeAb group. Pressure overload could increase vascular tolerance to I/R injury, but also trigger severe global ventricular fibrosis and results in atrial thrombus and pulmonary edema.

Current rodent models of ischemia/infarct or pressure-volume overload are not fully representative of human heart failure. We developed a new model of congestive heart failure (CHF) with both ischemic and stress injuries combined with fibrosis in the remote myocardium. Sprague-Dawley male rats were used. Ascending aortic banding (Ab) was performed to induce hypertrophy. Two months post-Ab, ischemia-reperfusion (I/R) injury was induced by ligating the left anterior descending (LAD) artery for 30 min. Permanent LAD ligation served as positive controls. A debanding (DeAb) procedure was performed after Ab or Ab + I/R to restore left ventricular (LV) loading properties. Cardiac function was assessed by echocardiography and in vivo hemodynamic analysis. Myocardial infarction (MI) size and myocardial fibrosis were assessed. LV hypertrophy was observed 4 mo post-Ab; however, systolic function was preserved. LV hypertrophy regressed within 1 mo after DeAb. I/R for 2 mo induced a small to moderate MI with mild impairment of LV function. Permanent LAD ligation for 2 mo induced large MI and significant cardiac dysfunction. Ab for 2 mo followed by I/R for 2 mo (Ab + I/R) resulted in moderate MI with significantly reduced ejection fraction (EF). DeAb post Ab + I/R to reduce afterload could not restore cardiac function. Perivascular fibrosis in remote myocardium after Ab + I/R + DeAb was associated with decreased cardiac function. We conclude that Ab plus I/R injury with aortic DeAb represents a novel model of CHF with increased fibrosis in remote myocardium. This model will allow the investigation of vascular and fibrotic mechanisms in CHF characterized by low EF, dilated LV, moderate infarction, near-normal aortic diameter, and reperfused coronary arteries.

Recently, cultured human adult skin cells were reprogrammed to induced pluripotent stem (iPS) cells, which have characteristics similar to human embryonic stem (hES) cells. Patient-derived iPS cells offer genetic and immunologic advantages for cell and tissue replacement or engineering. The efficiency of generating human iPS cells has been very low; therefore an easily and efficiently reprogrammed cell type is highly desired. Here, we demonstrate that terminally differentiated human amniotic fluid (AF) skin cells provide an accessible source for efficiently generating abundant-induced pluripotent stem (AF-iPS) cells. By induction of pluripotency with the transcription factor quartet (OCT3/4, SOX2, KLF4, and c-MYC) the terminally differentiated, cultured AF skin cells formed iPS colonies approximately twice as fast and yielded nearly a two-hundred percent increase in number, compared to cultured adult skin cells. AF-iPS cells were identical to hES cells for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, telomerase activity, in vitro and in vivo differentiation into the three germ layers and for their capacity to form embryoid bodies (EBs) and teratomas. Our findings provide a biological interesting conclusion that these fetal AF cells are more rapidly, easily, and efficiently reprogrammed to pluripotency than neonatal and adult cells. AF-iPS cells may have a “young,” more embryonic like epigenetic background, which may facilitate and accelerate pluripotency. The ability to efficiently and rapidly reprogram terminally differentiated AF skin cells and generate induced pluripotent stem cells provides an abundant iPS cell source for various basic studies and a potential for future patient-specific personalized therapies.