Development of Multifunctional Overhauser-enhanced Magnetic Rresonance Imaging for Concurrent in Vivo Mapping of Tumor Interstitial Oxygenation, Acidosis and Inorganic Phosphate Concentration

Tumor oxygenation (pO2), acidosis (pH) and interstitial inorganic phosphate concentration (Pi) are important parameters of the malignant behavior of cancer. A noninvasive procedure that enables visualization of these parameters may provide unique information about mechanisms of tumor pathophysiology and provide clues to new treatment targets. In this research, we present a multiparametric imaging method allowing for concurrent mapping of pH, spin probe concentration, pO2, and Pi using a single contrast agent and Overhauser-enhanced magnetic resonance imaging technique. The developed approach was applied to concurrent multifunctional imaging in phantom samples and in vivo in a mouse model of breast cancer. Tumor tissues showed higher heterogeneity of the distributions of the parameters compared with normal mammary gland and demonstrated the areas of significant acidosis, hypoxia, and elevated Pi content

Interstitial Inorganic Phosphate as a Tumor Microenvironment Marker for Tumor Progression

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO2), extracellular acidosis (pHe), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivo concurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO2, pHe and tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the “growth rate hypothesis” may underline its potential role in tumorigenesis and tumor progression

Interstitial Inorganic Phosphate as a Tumor Microenvironment Marker for Tumor Progression

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO2), extracellular acidosis (pHe), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivoconcurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO2, pHe and tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the “growth rate hypothesis” may underline its potential role in tumorigenesis and tumor progression

Cell Competition Boosts Clonal Evolution and Hypoxic Selection in Cancer

The comparison of fitness between cells leads to the elimination of less competent cells in the presence of more competent neighbors via cell competition (CC). This phenomenon has been linked with several cancer-related genes and thus may play an important role in cancer. Various processes are involved in the regulation of tumor initiation and growth, including tumor hypoxia, clonal stem cell selection, and immune cell response, all of which have been recently shown to have a potential connection with the mechanisms involved in CC. This review aims to unravel the relation between these processes and competitive cell interactions and how this affects disease progression

Macrophage and neutrophil interactions in the pancreatic tumor microenvironment drive the pathogenesis of pancreatic cancer

Despite modest improvements in survival in recent years, pancreatic adenocarcinoma remains a deadly disease with a 5-year survival rate of only 9%. These poor outcomes are driven by failure of early detection, treatment resistance, and propensity for early metastatic spread. Uncovering innovative therapeutic modalities to target the resistance mechanisms that make pancreatic cancer largely incurable are urgently needed. In this review, we discuss the immune composition of pancreatic tumors, including the counterintuitive fact that there is a significant inflammatory immune infiltrate in pancreatic cancer yet anti-tumor mechanisms are subverted and immune behaviors are suppressed. Here, we emphasize how immune cell interactions generate tumor progression and treatment resistance. We narrow in on tumor macrophage (TAM) spatial arrangement, polarity/function, recruitment, and origin to introduce a concept where interactions with tumor neutrophils (TAN) perpetuate the microenvironment. The sequelae of macrophage and neutrophil activities contributes to tumor remodeling, fibrosis, hypoxia, and progression. We also discuss immune mechanisms driving resistance to standard of care modalities. Finally, we describe a cadre of treatment targets, including those intended to overcome TAM and TAN recruitment and function, to circumvent barriers presented by immune infiltration in pancreatic adenocarcinoma

Analysis of single nuclear chromatin accessibility reveals unique myeloid populations in human pancreatic ductal adenocarcinoma

Background: A better understanding of the pancreatic ductal adenocarcinoma (PDAC) immune microenvironment is critical to developing new treatments and improving outcomes. Myeloid cells are of particular importance for PDAC progression; however, the presence of heterogenous subsets with different ontogeny and impact, along with some fluidity between them, (infiltrating monocytes vs. tissue‐resident macrophages; M1 vs. M2) makes characterisation of myeloid populations challenging. Recent advances in single cell sequencing technology provide tools for characterisation of immune cell infiltrates, and open chromatin provides source and function data for myeloid cells to assist in more comprehensive characterisation. Thus, we explore single nuclear assay for transposase accessible chromatin (ATAC) sequencing (snATAC‐Seq), a method to analyse open gene promoters and transcription factor binding, as an important means for discerning the myeloid composition in human PDAC tumours. Methods: Frozen pancreatic tissues (benign or PDAC) were prepared for snATAC‐Seq using 10× Chromium technology. Signac was used for preliminary analysis, clustering and differentially accessible chromatin region identification. The genes annotated in promoter regions were used for Gene Ontology (GO) enrichment and cell type annotation. Gene signatures were used for survival analysis with The Cancer Genome Atlas (TCGA)‐pancreatic adenocarcinoma (PAAD) dataset. Results: Myeloid cell transcription factor activities were higher in tumour than benign pancreatic samples, enabling us to further stratify tumour myeloid populations. Subcluster analysis revealed eight distinct myeloid populations. GO enrichment demonstrated unique functions for myeloid populations, including interleukin‐1b signalling (recruited monocytes) and intracellular protein transport (dendritic cells). The identified gene signature for dendritic cells influenced survival (hazard ratio = .63, p = .03) in the TCGA‐PAAD dataset, which was unique to PDAC. Conclusions: These data suggest snATAC‐Seq as a method for analysis of frozen human pancreatic tissues to distinguish myeloid populations. An improved understanding of myeloid cell heterogeneity and function is important for developing new treatment targets in PDAC

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation

The Underexplored Landscape of Hypoxia-Inducible Factor 2 Alpha and Potential Roles in Tumor Macrophages: A Review

Low tissue oxygenation, termed hypoxia, is a characteristic of solid tumors with negative consequences. Tumor-associated macrophages (TAMs) accumulate in hypoxic tumor regions and correlate with worse outcomes in cancer patients across several tumor types. Thus, the molecular mechanism in which macrophages respond to low oxygen tension has been increasingly investigated in the last decade. Hypoxia stabilizes a group of hypoxia-inducible transcription factors (HIFs) reported to drive transcriptional programs involved in cell survival, metabolism, and angiogenesis. Though both tumor macrophage HIF-1α and HIF-2α correlate with unfavorable tumor microenvironments, most research focuses on HIF-1α as the master regulator of hypoxia signaling, because HIF-1α expression was originally identified in several cancer types and correlates with worse outcome in cancer patients. The relative contribution of each HIFα subunit to cell phenotypes is poorly understood especially in TAMs. Once thought to have overlapping roles, recent investigation of macrophage HIF-2α has demonstrated a diverse function from HIF-1α. Little work has been published on the differential role of hypoxia-dependent macrophage HIF-2α when compared to HIF-1α in the context of tumor biology. This review highlights cellular HIF-2α functions and emphasizes the gap in research investigating oxygen-dependent functions of tumor macrophage HIF-2α

Monocytes and Macrophages Regulate Immunity through Dynamic Networks of Survival and Cell Death

Monocytes and macrophages are central cells of the innate immune system, responsible for defending against diverse pathogens. While they originate from a common myeloid precursor and share functions in innate immunity, each has a very distinct life span finely tuned by the apoptotic caspases. Normally, circulating monocytes are short-lived and undergo spontaneous apoptosis on a daily basis. Macrophages, however, have a longer life span. In chronic inflammatory diseases and, as recently recognized, in the tumor microenvironment, the inhibition of the apoptotic program promotes monocyte survival contributing to the accumulation of macrophages and the persistence of an inflammatory milieu. A complex network of differentiation factors and inflammatory stimuli determine monocyte/macrophage life span by blocking the apoptotic pathway and activating a myriad of survival pathways. Our understanding of apoptosis has flourished over the last decade, and its relevance in the regulation of the immune system is now indisputable. Nevertheless, how the complicated networks of survival and apoptotic regulators are integrated to determine cellular life span remains elusive. This review summarizes the contribution of the caspases and their regulators in monocyte/macrophage cell fate and discusses how these molecules orchestrate the initiation, maintenance, and resolution of inflammation. More provocatively, we discuss possible strategies to control inflammation by manipulating leukocyte life span

Immune Cell Modulation of the Extracellular Matrix Contributes to the Pathogenesis of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a five-year survival rate of only 9%. PDAC is characterized by a dense, fibrotic stroma composed of extracellular matrix (ECM) proteins. This desmoplastic stroma is a hallmark of PDAC, representing a significant physical barrier that is immunosuppressive and obstructs penetration of cytotoxic chemotherapy agents into the tumor microenvironment (TME). Additionally, dense ECM promotes hypoxia, making tumor cells refractive to radiation therapy and alters their metabolism, thereby supporting proliferation and survival. In this review, we outline the significant contribution of fibrosis to the pathogenesis of pancreatic cancer, with a focus on the cross talk between immune cells and pancreatic stellate cells that contribute to ECM deposition. We emphasize the cellular mechanisms by which neutrophils and macrophages, specifically, modulate the ECM in favor of PDAC-progression. Furthermore, we investigate how activated stellate cells and ECM influence immune cells and promote immunosuppression in PDAC. Finally, we summarize therapeutic strategies that target the stroma and hinder immune cell promotion of fibrogenesis, which have unfortunately led to mixed results. An enhanced understanding of the complex interactions between the pancreatic tumor ECM and immune cells may uncover novel treatment strategies that are desperately needed for this devastating disease