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Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion, including the downregulation of HLA-I, potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet, the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune, and vasculature components across 323 fields of view. Single-cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core fields of view of responders. Integrating single-cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the tumor-centric cellular neighborhood (TCCN) based on anatomic subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytic framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy.Significance:Findings from this work propose a novel approach to resolving clinical heterogeneity of the TIME by objectively quantifying the cellular interactions occurring in metastatic melanoma lymph node tissue utilizing multiplex immunofluorescence. This study provides an analytic and biologically derived unit of measure, the TCCN which is customizable for studying critical paracrine interactions within spatially preserved tissue of various cancers and across the spectrum of multiplex imaging modalities.

Antibody reagents and validation for MxIF panel design

Summary data of FOV distribution, single cells segmented, classified and clinical response to therapy

Virtual H&E images for whole excisional lymph node biopsies from each clinical case

Supplementary Figure 1. Of 16 patients (7 with and 9 without germination) with available data, 11 (69%) had a decrease in tumor density (-4% to -476%). The most robust decreases in density were observed in patients with germination. Supplementary Figure 2. Tumor antigen specific T-cell responses after C. novyi injection. Supplementary Figure 3. Infiltration in the injected and non-injected tumors.

Purpose:Intratumorally injected Clostridium novyi-NT (nontoxic; lacking the alpha toxin), an attenuated strain of C. novyi, replicates within hypoxic tumor regions resulting in tumor-confined cell lysis and inflammatory response in animals, which warrants clinical investigation.Patients and Methods:This first-in-human study (NCT01924689) enrolled patients with injectable, treatment-refractory solid tumors to receive a single intratumoral injection of C. novyi-NT across 6 dose cohorts (1 × 104 to 3 × 106 spores, 3+3 dose-escalation design) to determine dose-limiting toxicities (DLT), and the maximum tolerated dose.Results:Among 24 patients, a single intratumoral injection of C. novyi-NT led to bacterial spores germination and the resultant lysis of injected tumor masses in 10 patients (42%) across all doses. The cohort 5 dose (1 × 106 spores) was defined as the maximum tolerated dose; DLTs were grade 4 sepsis (n = 2) and grade 4 gas gangrene (n = 1), all occurring in three patients with injected tumors >8 cm. Other treatment-related grade ≥3 toxicities included pathologic fracture (n = 1), limb abscess (n = 1), soft-tissue infection (n = 1), respiratory insufficiency (n = 1), and rash (n = 1), which occurred across four patients. Of 22 evaluable patients, nine (41%) had a decrease in size of the injected tumor and 19 (86%) had stable disease as the best overall response in injected and noninjected lesions combined. C. novyi-NT injection elicited a transient systemic cytokine response and enhanced systemic tumor-specific T-cell responses.Conclusions:Single intratumoral injection of C. novyi-NT is feasible. Toxicities can be significant but manageable. Signals of antitumor activity and the host immune response support additional studies of C. novyi-NT in humans.

Supplementary Legend

Purpose: Immunotherapeutic modalities are commonly used for treatment of patients with melanoma. The therapeutic success in preclinical models has not yielded the expected clinical results. To understand this discrepancy, we attempted to define immune homeostasis of 209 patients with melanoma across stages of disease relative to normal controls.Experimental Design: Peripheral blood mononuclear cells (PBMC) and plasma were collected from patients and healthy donors. PBMC were analyzed for frequencies of natural killer, dendritic, and T cells and their functional status. Matched plasma samples were analyzed for the concentrations of 27 cytokines, chemokines, and growth factors. RNA was isolated from 24 metastatic melanoma tumor biopsies and profiled by microarray analysis.Results: The frequency of natural killer, T, and dendritic cells in patients does not significantly change across stages of melanoma. However, plasma concentrations of Th2 cytokines [interleukin (IL)-4, IL-5, IL-10, and IL-13] in tumor-bearing patients were significantly higher than those with resected melanoma. Expression array analysis of metastatic melanoma revealed that the malignant melanocytes were not the source of the Th2 cytokines but did highly up-regulate vascular endothelial growth factor (VEGF) transcripts, consistent with plasma VEGF concentrations. In vitro VEGF exposure of normal PBMC lead to repolarization from Th1 to Th2 emulating the state of metastatic melanoma.Conclusions: Patients with metastatic melanoma exist in a state of Th2-mediated “chronic inflammation” as a result of at least VEGF overproduction by malignant tumors. These data support prior observations regarding the effect of VEGF on immune cell function and suggests consideration of VEGF inhibitors in future cancer immunotherapy clinical studies in metastatic melanoma.

Supplementary Table S1 from Evidence of Systemic Th2-Driven Chronic Inflammation in Patients with Metastatic Melanoma

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PDF file - 1.6MB, Confocal microscopy and western blot results showing inhibition of autophagy and increase in chemotherapy-induced cancer cell death by specific inhibition of VEGF-C/NRP-2 axis

Background Recent clinical advances in cancer immuno-therapeutics underscore the need for improved understanding of the complex relationship between cancer and the multiple, multi-functional, inter-dependent, cellular and humoral mediators/regulators of the human immune system. This interdisciplinary effort exploits engineering analysis methods utilized to investigate anomalous physical system behaviors to explore immune system behaviors. Cancer Immune Control Dynamics (CICD), a systems analysis approach, attempts to identify differences between systemic immune homeostasis of 27 healthy volunteers versus 14 patients with metastatic malignant melanoma based on daily serial measurements of conventional peripheral blood biomarkers (15 cell subsets, 35 cytokines). The modeling strategy applies engineering control theory to analyze an individual’s immune system based on the biomarkers’ dynamic non-linear oscillatory behaviors. The reverse engineering analysis uses a Singular Value Decomposition (SVD) algorithm to solve the inverse problem and identify a solution profile of the active biomarker relationships. Herein, 28,605 biologically possible biomarker interactions are modeled by a set of matrix equations creating a system interaction model. CICD quantifies the model with a participant’s biomarker data then computationally solves it to measure each relationship’s activity allowing a visualization of the individual’s current state of immunity. Results CICD results provide initial evidence that this model-based analysis is consistent with identified roles of biomarkers in systemic immunity of cancer patients versus that of healthy volunteers. The mathematical computations alone identified a plausible network of immune cells, including T cells, natural killer (NK) cells, monocytes, and dendritic cells (DC) with cytokines MCP-1 [CXCL2], IP-10 [CXCL10], and IL-8 that play a role in sustaining the state of immunity in advanced cancer. Conclusions With CICD modeling capabilities, the complexity of the immune system is mathematically quantified through thousands of possible interactions between multiple biomarkers. Therefore, the overall state of an individual’s immune system regardless of clinical status, is modeled as reflected in their blood samples. It is anticipated that CICD-based capabilities will provide tools to specifically address cancer and treatment modulated (immune checkpoint inhibitors) parameters of human immunity, revealing clinically relevant biological interactions.

Purpose: Intratumorally injected Clostridium novyi-NT (nontoxic; lacking the alpha toxin), an attenuated strain of C. novyi, replicates within hypoxic tumor regions resulting in tumor-confined cell lysis and inflammatory response in animals, which warrants clinical investigation. Patients and Methods: This first-in-human study (NCT01924689) enrolled patients with injectable, treatment-refractory solid tumors to receive a single intratumoral injection of C. novyi-NT across 6 dose cohorts (1 × 104 to 3 × 106 spores, 3+3 dose-escalation design) to determine dose-limiting toxicities (DLT), and the maximum tolerated dose. Results: Among 24 patients, a single intratumoral injection of C. novyi-NT led to bacterial spores germination and the resultant lysis of injected tumor masses in 10 patients (42%) across all doses. The cohort 5 dose (1 × 106 spores) was defined as the maximum tolerated dose; DLTs were grade 4 sepsis (n = 2) and grade 4 gas gangrene (n = 1), all occurring in three patients with injected tumors >8 cm. Other treatment-related grade ≥3 toxicities included pathologic fracture (n = 1), limb abscess (n = 1), soft-tissue infection (n = 1), respiratory insufficiency (n = 1), and rash (n = 1), which occurred across four patients. Of 22 evaluable patients, nine (41%) had a decrease in size of the injected tumor and 19 (86%) had stable disease as the best overall response in injected and noninjected lesions combined. C. novyi-NT injection elicited a transient systemic cytokine response and enhanced systemic tumor-specific T-cell responses. Conclusions: Single intratumoral injection of C. novyi-NT is feasible. Toxicities can be significant but manageable. Signals of antitumor activity and the host immune response support additional studies of C. novyi-NT in humans.

The placenta utilizes many mechanisms to protect the haploidentical fetus from recognition by the maternal immune system. However, in cases of villitis of unknown etiology (VUE), maternal lymphocytes gain access into the placenta, causing significant health risks for the fetus. Evidence suggests that VUE is a rejection response between the mother and the haploidentical fetus. Therefore, we profiled human leukocyte antigen (HLA), an important predictor of transplant rejection, in VUE using placental tissue from ten patients with VUE and ten gestational age matched controls. Placentas were stained using novel multiplexed immunofluorescence (MxIF) to investigate morphology and HLA classes I and II. Gene expression was evaluated by microarray, and where available, tissue typing of mother/baby pairs was completed to determine HLA type. MxIF demonstrated strong CD8+ T cell infiltration and HLA class I staining both the distal and stem villi of VUE placentas. Compared to controls, VUE cases had significantly higher expression of HLA class II mRNA and pathway analysis demonstrated that 40% of the differentially expressed genes in VUE are related to tissue rejection. The data suggest that VUE resembles a rejection response between the mother and the fetus. It remains unknown what initiates immune recognition and why some mothers appear to be at higher risk for developing this condition than others. Understanding this etiology will be critical for developing effective interventions or prevention strategies during pregnancy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s43032-019-00101-9) contains supplementary material, which is available to authorized users.

Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion, including the downregulation of HLA-I, potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet, the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune, and vasculature components across 323 fields of view. Single-cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core fields of view of responders. Integrating single-cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the tumor-centric cellular neighborhood (TCCN) based on anatomic subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytic framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy. Significance: Findings from this work propose a novel approach to resolving clinical heterogeneity of the TIME by objectively quantifying the cellular interactions occurring in metastatic melanoma lymph node tissue utilizing multiplex immunofluorescence. This study provides an analytic and biologically derived unit of measure, the TCCN which is customizable for studying critical paracrine interactions within spatially preserved tissue of various cancers and across the spectrum of multiplex imaging modalities.

The Center for Disease Control and Prevention ranks diabetes mellitus (DM) as the seventh leading cause of death in the USA. The most prevalent forms of DM include Type 2 DM, Type 1 DM, and gestational diabetes mellitus (GDM). While the acute problem of diabetic hyperglycemia can be clinically managed through dietary control and lifestyle changes or pharmacological intervention with oral medications or insulin, long-term complications of the disease are associated with significant morbidity and mortality. These long-term complications involve nearly all organ systems of the body and share common pathologies associated with endothelial cell abnormalities. To better understand the molecular mechanisms underlying DM as related to future long-term complications following hyperglycemia, we have undertaken a study to determine the frequency that GDM did or did not occur in the second pregnancy of women who experienced GDM in their first pregnancy between 2013 and 2018 at Mayo Clinic, Rochester, MN. Within the five-year period of the study, the results indicate that 7,330 women received obstetrical care for pregnancy during the study period. Of these, 150 developed GDM in their first pregnancy and of these, 42 (28%) had a second pregnancy. Of these 42 women, 20 again developed GDM and 22 did not develop GDM in their second pregnancy within the study period. Following the occurrence of GDM in the first pregnancy, the study (1) established the number of women with and without GDM in the second pregnancy and (2) confirmed the feasibility to study diabetic metabolic memory using maternal placental tissue from GDM women. These studies represent Phase I of a larger research project whose goal is to analyze epigenetic mechanisms underlying true diabetic metabolic memory using endothelial cells isolated from the maternal placenta of women with and without GDM as described in this article.

Background: Basic pitcher statistics have been used to assess performance in pitchers after injury or surgery without being validated. Even among healthy pitchers, the normal variability of these parameters has not yet been established. Purpose: To determine (1) the normal variability of basic and advanced pitcher statistics in healthy professional baseball pitchers and (2) the minimum pitches needed to predict these parameters. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Publicly available data from the MLB Statcast and PITCHf/x databases were used to analyze MLB pitchers during the 2015 and 2016 seasons who recorded a minimum of 100 innings without injury. Basic and advanced baseball pitcher statistics were analyzed. The variability of each parameter was assessed by computing the coefficient of variation (CV) between individual pitchers and across all pitchers. A CV 10 were generally considered inconsistent and unreliable. The minimum number of pitches needed to be followed for each variable was also analyzed. Results: A total of 118 pitchers, 55 baseball-specific statistical metrics (38 basic and 17 advanced), and 7.5 million pitches were included and analyzed. Of the 38 basic pitcher statistics, only fastball velocity demonstrated a CV Conclusion: We recommend against utilizing nonvalidated statistical measures to assess performance after injury, as they demonstrated unacceptably high variability even among healthy, noninjured professional baseball pitchers. It is our hope that this study will serve as the foundation for the identification and implementation of validated pitcher-dependent statistical measures that can be used to assess return-to-play performance after injury in the future.

Intratumorally injectedThis first-in-human study (NCT01924689) enrolled patients with injectable, treatment-refractory solid tumors to receive a single intratumoral injection ofAmong 24 patients, a single intratumoral injection ofSingle intratumoral injection of

Background Development of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. Methods We have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα+) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7Raf-1) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database. Results In this study, we identified an association between NOTCH3 expression and development of metastases in ERα+ and TNBC models. ERα+ breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44low/CD24high/ERαhigh phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens. Conclusions These findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer. Electronic supplementary material The online version of this article (10.1186/s13058-018-1020-0) contains supplementary material, which is available to authorized users.

In patients with metastatic melanoma, high blood levels of galectin-9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin-9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin-9 with macrophages. We aimed to determine whether galectin-9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor-supportive. It was determined that incubation of CD68+ macrophages with galectin-9 or anti-CD206 blocked target binding and that both CD206 and galectin-9 were detected by immunoprecipitation of cell lysates. CD206 and galectin-9 had a binding affinity of 2.8 × 10-7 m. Galectin-9 causes CD206+ macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP-1), but less macrophage-derived chemokine (MDC). Galectin-9 had no effect on classical monocyte subsets, but caused expansion of the non-classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin-9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin-9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti-tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Women diagnosed with cutaneous melanoma have a survival advantage compared to men, which has been hypothesized to be due to difference in behavior and/or biology (sex hormones). It remains controversial whether this advantage is dependent on age or stage of disease. We sought to compare melanoma‐specific survival between females in pre, peri, and postmenopausal age groups to males in the same age group, adjusting for stage of disease. This is a retrospective population‐based cohort study using the Surveillance, Epidemiology, and End Results (SEER) database. Patients diagnosed from 1 January 1992 through 31 January 2011 with primary invasive cutaneous melanoma were included in our cohort. Melanoma‐specific survival was the main outcome studied. Of the 106,511 subjects that were included, 45% were female. Females in all age groups (18–45, 46–54, and ≥55) with localized and regional disease, were less likely to die from melanoma compared to males in the same age group. Among patients with localized and regional disease, the relative risk of death due to melanoma increased with advancing age at diagnosis; this increase was more pronounced among females than males. In contrast, we observed no female survival advantage among patients with distant disease and no effect of age on relative risk of death from melanoma. Females with localized and regional melanoma have a decreased risk of death compared to males within all age groups. Our data show no differences in survival between men and women with metastatic melanoma, indicating that the influence of sex on survival is limited to early stage disease but not confined to pre or perimenopausal age groups.

Galectin-9, a β-galactoside-binding protein, is defined as a negative regulator of T helper 1 (Th1) immune responses, favoring Th2 bias. Systemic immunity in patients with metastatic melanoma is predominantly Th2 biased. We hypothesized that galectin-9 can modulate systemic immunity toward Th2 polarization in patients with advanced melanoma. The presence or concentration of galectin-9 was assessed in tumors and plasma, in patients with metastatic melanoma. The immunomodulatory function of galectin-9 was determined by exposing human peripheral blood mononuclear cells to galectin-9 in vitro. Galectin-9 was expressed in 57% of tumors and was significantly (3.6-fold) increased in the plasma of patients with advanced melanoma compared with healthy controls (P

e15184 Background: Clinical responses to anti-PD1 immunotherapy in patients with metastatic melanoma (MM) remain challenging to predict. This clinical heterogeneity is also reflected in the tumor-immune microenvironment among patients and within a single tumor lesion. With the emergence of multiplex imaging platforms, defining complex phenotypes at single cell resolution has become possible. Here, we sought to objectively quantify paracrine tumor-immune interactions contributing to the variable clinical responses observed in patients receiving anti-PD1 therapy. Methods: Excisional lymph node (LN) biopsies were obtained from treatment-naïve patients with MM who underwent subsequent anti-PD1 therapy. A single 5µm section of LN tissue was used to assess a 42 analyte panel by multiplex immunofluorescence. From 30 LN samples, 418 fields of view (FOVs) were selected resulting in 14,360 high-resolution images of 4 anatomical subregions: tumor core, tumor-immune interface, tumor infiltrate and adjacent immune stroma. Following image processing, we developed an adaptive classification for tumor-centric cellular neighborhoods (TCCN) to identify and quantify critical paracrine interactions within the tumor-immune microenvironment. Results: Stratification based on responsiveness to anti-PD1 therapy resulted in 4 complete responders (CR) and 12 non-responders (NR) at 12-week follow-up. From 126 FOVs, we defined the cellular composition of 197,865 TCCN across patients based on clinical response and LN subregions. Overall, the percentage of TCCN devoid of any T cells, B cells or macrophages was significantly higher in NR compared to CR irrespective of subregion. However, other markers differentiated TCCN based on subregion. Specifically in CR, tumor core regions were enriched for CD8 T cells, while enrichment for B cells and endothelial cells was demonstrated at the tumor-immune interface. Strikingly, tumor infiltrate regions demonstrate robust immune reactivity with enrichment for M1 polarized macrophages, NK cells and B cells in CR compared to NR. Complete data from the 30 patient cohort across 418 FOVs will be presented. Conclusions: Taken together, this data suggests cellular composition of TCCN across subregions of the LN is dynamic within a single metastatic site. In this small cohort, we introduce a formalized stratification to quantify and classify critical paracrine interactions within the immune-tumor microenvironment with the potential to inform clinical responsiveness to therapy.

Background Attenuated measles virus (MV) strains are promising agents currently being tested against solid tumors or hematologic malignancies in ongoing phase I and II clinical trials; factors determining oncolytic virotherapy success remain poorly understood, however. Methods We performed RNA sequencing and gene set enrichment analysis to identify pathways differentially activated in MV-resistant (n = 3) and -permissive (n = 2) tumors derived from resected human glioblastoma (GBM) specimens and propagated as xenografts (PDX). Using a unique gene signature we identified, we generated a diagonal linear discriminant analysis (DLDA) classification algorithm to predict MV responders and nonresponders, which was validated in additional randomly selected GBM and ovarian cancer PDX and 10 GBM patients treated with MV in a phase I trial. GBM PDX lines were also treated with the US Food and Drug Administration-approved JAK inhibitor, ruxolitinib, for 48 hours prior to MV infection and virus production, STAT1/3 signaling and interferon stimulated gene expression was assessed. All statistical tests were two-sided. Results Constitutive interferon pathway activation, as reflected in the DLDA algorithm, was identified as the key determinant for MV replication, independent of virus receptor expression, in MV-permissive and -resistant GBM PDXs. Using these lines as the training data for the DLDA algorithm, we confirmed the accuracy of our algorithm in predicting MV response in randomly selected GBM PDX ovarian cancer PDXs. Using the DLDA prediction algorithm, we demonstrate that virus replication in patient tumors is inversely correlated with expression of this resistance gene signature (ρ = -0.717, P = .03). In vitro inhibition of the interferon response pathway with the JAK inhibitor ruxolitinib was able to overcome resistance and increase virus production (1000-fold, P = .03) in GBM PDX lines. Conclusions These findings document a key mechanism of tumor resistance to oncolytic MV therapy and describe for the first time the development of a prediction algorithm to preselect for oncolytic treatment or combinatorial strategies.

Diabetes mellitus (DM) is classified as a disease of metabolic dysregulation predicted to affect over 400 million individuals world-wide by 2030. The debilitating aspects of this disease are the long term complications involving microvascular and macrovascular pathologies. These long term complications are related to the clinical phenomenon of metabolic memory (MM) that is defined as the persistence of diabetic complications even after glycemic control has been pharmacologically achieved. The persistent nature of MM has invoked involvement of epigenetic processes. Current research with the DM/MM zebrafish model as described in this review as well as human and mammalian studies has established that changes in DNA methylation patterns appear to contribute to tissue dysfunctions associated with DM. This review will describe studies on an adult zebrafish model of type I diabetes mellitus that allows analysis of both the hyperglycemic (HG or DM) phase and MM phase of the disease. The review will discuss the model in regards to: 1) its hyperglycemic phase, 2) its MM phase, 3) biochemical õpathways underlying changes in DNA methylation patterns observed in the model, 4) loci specific changes in DNA methylation patterns, and 5) strengths of the adult zebrafish model as compared to other MM animal models.

Evidence suggests that immunological response in chronic inflammation is dynamic, oscillating between active immunity and tolerance. We hypothesized that a similar dynamic exists in melanoma and administration of therapy during a unique phase of such oscillation could impact clinical outcome. Patients with metastatic melanoma eligible to undergo temozolomide underwent serial measurements of C-reactive protein (CRP) and immune biomarkers every 2-3 days for 2 weeks before starting therapy. Treatment was initiated prior to the estimated next CRP peak, or on day 14 post-registration if a peak was not identified. Time profiles of measured biomarkers were analyzed by fitting serially measured data points to 9 mathematical functions and were correlated to time of therapy and outcome. Data suggested that metastatic melanoma patients exhibit a dynamic immune response. The fluctuation of several biomarkers fitted cosine functions with periods which were multiples of 3-4 days. Chemotherapy delivery during a unique phase of this cycle seemed to correlate with improved response. Individualized conventional chemotherapy delivery by synchronizing treatment with pre-existing patient-specific biorhythms may improve clinical outcomes in metastatic melanoma.

The fundamental and critical functions of the ECM to metazoans explain why we see its appearance in ancient Taxa such as Cnidaria that represents one of the earliest groups with a defined tissue organization and an ECM A model system for understanding the mechanisms involved in Cell ECM interactions during developmental processes is the Cnidarian Hydra diverged some million years ago whose use in developmental biology dates back to the studies of Trembley in As discussed in this review ECM does not stand alone but rather is part of a complicated system that we refer to as the Extracellular matrix Integrin Matrix Metalloproteinase Network ECM Integrin MMP Network This network not only provides structural support for tissues but also functions as a signal transduction platform that regulates cell physiological and developmental processes The purpose of the review is to show the highly conserved nature of the ECM Integrin MMP Network throughout the metazoans and does so by comparing what we know of the vertebrate network to that of the Hydra network The review is organized to discuss A nbsp nbsp nbsp nbsp nbsp nbsp The initial studies related to the structure and function of Hydra ECM prior to B nbsp nbsp nbsp nbsp nbsp nbsp A discussion of the Hydra ECM project that utilized a convergent biochemical cellular and molecular approach to the study of Hydra ECM and its associated molecules after C nbsp nbsp nbsp nbsp nbsp nbsp The composition of the vertebrate network and D nbsp nbsp nbsp nbsp nbsp A comparative analysis of the vertebrate network to the Hydra network based on the most current NCBI genomic and EST databases

A systemic analysis of the tumor-immune interactions within the heterogeneous tumor microenvironment is of particular importance for understanding the antitumor immune response. We used multiplexed immunofluorescence to elucidate cellular spatial interactions and T-cell infiltrations in metastatic melanoma tumor microenvironment. We developed two novel computational approaches that enable infiltration clustering and single cell analysis-cell aggregate algorithm and cell neighborhood analysis algorithm-to reveal and to compare the spatial distribution of various immune cells relative to tumor cell in sub-anatomic tumor microenvironment areas. We showed that the heterogeneous tumor human leukocyte antigen-1 expressions differently affect the magnitude of cytotoxic T-cell infiltration and the distributions of CD20+ B cells and CD4+FOXP3+ regulatory T cells within and outside of T-cell infiltrated tumor areas. In a cohort of 166 stage III melanoma samples, high tumor human leukocyte antigen-1 expression is required but not sufficient for high T-cell infiltration, with significantly improved overall survival. Our results demonstrate that tumor cells with heterogeneous properties are associated with differential but predictable distributions of immune cells within heterogeneous tumor microenvironment with various biological features and impacts on clinical outcomes. It establishes tools necessary for systematic analysis of the tumor microenvironment, allowing the elucidation of the "homogeneous patterns" within the heterogeneous tumor microenvironment.

e21058 Background: Recent clinical advances in cancer immuno-therapeutics underscore the need for improved understanding of the complex relationship between cancer and the multiple, multi-functional, inter-dependent, cellular and humoral mediators/regulators of the human immune system. This will lead to improvements in the efficacy of existing immune therapies (patient selection; drug combinations) and insight into new therapeutic targets that may significantly reduce the time to new therapeutics discoveries capable of meaningful clinical impacts. Methods: A systems analysis approach (Cancer Immune Control Dynamics, CICD) attempts to identify the differences between systemic immune homeostasis of healthy volunteers versus patients with metastatic malignant melanoma based on serial measurements of a set of conventional peripheral blood biomarkers of immune function (15 cell subsets and 35 cytokines) assessed daily over 7-10 days. The modeling strategy takes aerospace engineering control theory and system identification algorithms to analyze the state of an individual’s immune system based on the biomarkers’ dynamic non-linear oscillatory behaviors. The analysis framework builds on the classical predator-prey equations commonly used to model biological system dynamics. Tens of thousands of biologically possible biomarker interactions are modeled by a set of matrix equations that creates a system interaction model. CICD quantifies the system equations with biomarker values and solves it to measure the interactions. Results: Quantification of thousands of unique biomarker relationships per participant enables classification based on their immune profile rather than their clinical diagnosis and a basis for a visualization of a person’s immunity state. Conclusions: Results provide initial evidence that a model-based analysis of serially collected biomarker data can lead to biological insights into human immunity, of potential benefit in clinical decisions. It is anticipated that CICD-based capabilities will provide tools to specifically address cancer and treatment modulated (immune checkpoint inhibitors) parameters of human immunity, revealing clinically relevant biological interactions.

Bacteriolytic strategies offer unique advantages to combat a broad range of cancers often refractive to conventional chemotherapies and/or radiotherapies. C. novyi-NT is an attenuated strain of Clostridium novyi, a spore-forming, gram-positive, obligate anaerobe that lacks a lethal alpha- toxin, expressed by the parental strain. When administered intravenously or intratumorally with percutaneous injection, C. novyi-NT colonizes and replicates within the hypoxic region of the tumors, eliciting robust, tumor-confined cell lysis. In this first-in-man phase 1 study, patients with injectable treatment-refractory solid tumors received a single intratumoral injection of C. novyi-NT spores across 6 dose cohorts (spore concentrations of 104, 3x104, 105, 3x105, 106, 3x106) using a 3+3 trial design. The primary endpoints were to assess the safety profile, the dose limiting toxicity, and the maximum tolerated dose of C. novyi-NT. Key secondary endpoints included assessments of the preliminary antitumor activity of the injected tumor, an overall response evaluated by RECIST v. 1.1, and the host immune and inflammatory response to C. novyi-NT. Twenty-four patients were enrolled between November, 2013 and April, 2017. A single intratumoral injection of C. novyi-NT led to germination and resultant tumor lysis of injected tumor masses in 46% of patients across all dosing cohorts. The cohort 5 dose of 106 spores was defined as the maximum tolerated dose. Dose-limiting toxicities were grade 4 sepsis and grade 4 gas gangrene (n=1), all in patients with germination. In the 22 evaluable patients, 21 (95%) had stable disease (SD) as the best response for the injected lesion (tumor shrinkage of > 10% was observed in 21% of patients) and 19 (86%) had overall SD as the best response per RECIST 1.1. C. novyi-NT injection resulted in transient serum cytokine responses consistent with inflammasome activity, activation of innate immunity, tissue remodeling, and angiogenesis. Tumor antigen specific ELISPOT assays pre- and post- treatment documented enhanced secretion of IFNγ and TNFα by circulating T-cells, indicating improved systemic tumor specific T-cell responses. Multiparametric in situ immunostaining of core biopsies suggested improved immune cell infiltration in metastatic lesions. These early signs of improved antitumor activity in patients with advanced solid tumors have encouraged a new trial of C. novyi-NT in combination with immune checkpoint inhibitors. Citation Format: Filip Janku, Mrinal Gounder, Abdul Mohammad Pezeshki, Ravi Murthy, Andrea Wang-Gillam, Dale Shepard, David S. Hong, Sarina A. Piha-Paul, Anjali Raina, Alexey A. Leontovich, Gary DeCrescenzo, Brent L. Kreider, David Tung, Mary Varterasian, Halle H. Zhang, Khashayarsha Khazaie. First-in-man clinical trial of intratumoral injection of Clostridiumnovyi-NT spores in patients with treatment-refractory advanced solid tumors: Safety, activity, and immune responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A011.

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm–Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)–enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A 2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.

JAK-STAT is a rational drug target in myelofibrosis (MF) given its association with JAK2/MPL mutations and aberrant inflammatory cytokine expression. We conducted a Phase 1/2 trial of CYT387, a potent JAK1/2 inhibitor, in patients with high- or intermediate-risk primary or post-polycythemia vera/essential thrombocythemia MF. Pre-planned safety and efficacy analysis has been completed for the initial 60 patients. In the dose-escalation phase (n=21), the maximum-tolerated dose was 300 mg/day based on reversible grade 3 headache and asymptomatic hyperlipasemia. Twenty-one and 18 additional patients were accrued at two biologically effective doses, 300 mg/day and 150 mg/day, respectively. Anemia and spleen responses, per International Working Group criteria, were 59% and 48%, respectively. Among 33 patients who were red cell-transfused in the month prior to study entry, 70% achieved a minimum 12-week period without transfusions (range 4.7–>18.3 months). Most patients experienced constitutional symptoms improvement. Grade 3/4 adverse reactions included thrombocytopenia (32%), hyperlipasemia (5%), elevated liver transaminases (3%) and headache (3%). New-onset treatment-related peripheral neuropathy was observed in 22% of patients (sensory symptoms, grade 1). CYT387 is well tolerated and produces significant anemia, spleen and symptom responses in MF patients. Plasma cytokine and gene expression studies suggested a broad anticytokine drug effect.

As previously reported by our laboratory, streptozocin-induced diabetes mellitus (DM) in adult zebrafish results in an impairment of tissue regeneration as monitored by caudal fin regeneration. Following streptozocin withdrawal, a recovery phase occurs to reestablish euglycemia, via pancreatic beta-cell regeneration. However, DM-associated impaired fin regeneration continues indefinitely in the metabolic memory (MM) state, allowing for subsequent molecular analysis of the underlying mechanisms of MM. This study focuses on elucidating the molecular basis that explains the DM-associated impaired fin regeneration and why it persists into the MM state with the aim of better understanding MM. Using a combination of microarray analysis and bioinformatics approaches, our study found that of the 14,900 transcripts analyzed, aberrant expression of 71 genes relating to tissue developmental and regeneration processes were identified in DM fish and the altered expression of these 71 genes persisted in MM fish. Key regulatory genes of major development and signal transduction pathways were identified among this group of 71. The aberrant expression of key regulatory genes in the DM state that persist into the MM state provides a plausible explanation on how hyperglycemia induced impaired fin regeneration in the adult zebrafish DM/MM model.

A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in cancer cells. One pathway contributing to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from death. We have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which helps cancer cell survival following treatment. Inhibition of mTOR complex 1 activity by this axis is the underlying mechanism for the activation of autophagy. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1, that have previously been suggested to participate in autophagy and vesicular trafficking. Upregulation of WDFY-1 following VEGF-C or NRP-2 depletion contributes to cytotoxic drug-mediated cell death. Together, these data suggest a link between the VEGF-C/NRP-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress. Effective targeting of this pathway may lead to the development of new cancer therapies. Cancer Res; 73(1); 160–71. ©2012 AACR.

Skin cancer affects more individuals in the USA than any other malignancy and malignant melanoma is particularly deadly because of its metastatic potential. Melanoma has been recognized as one of the most immunogenic malignancies; therefore, understanding the mechanisms of tumor-immune interaction is key for developing more efficient treatments. As the tumor microenvironment shows an immunosuppressive action, immunotherapeutic agents promoting endogenous immune response to cancer have been tested (interleukin-2, anticytotoxic-T-lymphocyte-associated antigen 4, and antiprogrammed cell death protein 1 monoclonal antibodies) as well as combinations of cytotoxic chemotherapy agents and inhibitors of angiogenesis (taxol/carboplatin/avastin). However, clinical outcomes are variable, with only a minority of patients achieving durable complete responses. The variability of immune homeostasis, which may be more active or more tolerant at any given time, in cancer patients and the interaction of the immune system with the tumor could explain the inconsistency in clinical outcomes among these patients. Recently, the role of the lymphocyte-to-monocyte-ratio (LMR) in the peripheral blood has been investigated and has been proven to be an independent predictor of survival in different hematological malignancies and in solid tumors. In melanoma, our group has validated the significance of LMR as a predictor of relapse after resection of advanced melanoma. In this study, we examined the dynamics in the immune system of patients with advanced melanoma by performing serial multiday concentration measurements of cytokines and immune cell subsets in the peripheral blood. The analysis of outcomes of chemotherapy administration as related to LMR on the day of treatment initiation showed that progression-free survival was improved in the patients who received chemotherapy on the day when LMR was elevated.

Osteoarthritis (OA) is a disabling degenerative joint disease that prompts pain and has limited treatment options. To permit early diagnosis and treatment of OA, a high resolution mechanistic understanding of human chondrocytes in normal and diseased states is necessary. In this study, we assessed the biological effects of OA-related changes in the synovial microenvironment on chondrocytes embedded within anatomically intact cartilage from joints with different pathological grades by next generation RNA-sequencing (RNA-seq). We determined the transcriptome of primary articular chondrocytes derived from anatomically unaffected knees and ankles, as well as from joints affected by OA. The GALAXY bioinformatics platform was used to facilitate biological interpretations. Comparisons of patient samples by k-means, hierarchical clustering and principal component analyses together reveal that primary chondrocytes exhibit OA grade-related differences in gene expression, including genes involved in cell-adhesion, ECM production and immune response. We conclude that diseased synovial microenvironments in joints with different histopathological OA grades directly alter gene expression in chondrocytes. One ramification of this finding is that anatomically intact cartilage from OA joints is not an ideal source of healthy chondrocytes, nor should these specimens be used to generate a normal baseline for the molecular characterization of diseased joints.

Summary We have observed T helper type 2 (Th2) polarization of systemic immunity in patients with metastatic malignant melanoma. We hypothesized that similar changes in systemic immunity occur with ageing and may be permissive for the development of melanoma. We analysed the peripheral blood of 389 healthy blood donors. All subjects were profiled for peripheral blood T cell and B cell subsets, and 58 of these subjects were profiled for antigen-specific cytotoxic T cell subsets [cytomegalovirus (CMV), influenza and melanoma antigen recognized by T cells 1 (MART-1)]. Ninety-five separate healthy subjects underwent profiling of 42 plasma cytokines. Ageing was associated positively with CD4+CD294+ Th2 cells, and associated negatively with CD3+ T cells, cytotoxic T cells and T helper cells. Ageing was also associated negatively with CMV-, influenza- and MART-1-specific naive and CD8+ T cells. There were significant increases in plasma monocyte chemotactic protein 1 (MCP-1) (CCL1) and regulated upon activation normal T cell expressed and secreted (RANTES) (CCL5) with age. We observed differences in cytokine profiles between males and females; specifically, women had higher levels of sCD40L and PDGF-AA. In summary, we demonstrated in healthy blood donors that ageing was associated with an increase in cellular Th2 bias and a decline in total numbers of T cells. Additionally, there was an increase in MCP-1 and RANTES with ageing. Women had higher levels of sCD40L and PDGF-AA than men.