Toward a rational design of combination therapy in cancer.

By merging computational systems modeling and experimental approaches, we have uncovered treatments reprogramming pro-angiogenic monocytes present in breast tumor into immunologically potent cells capable of mediating an anti-tumor immune response. The unraveled pathways and ligands which underlie monocyte pro-angiogenic activity have a strong predictive value for breast cancer patient relapse - free survival

Social networks help to infer causality in the tumor microenvironment.

BACKGROUND: Networks have become a popular way to conceptualize a system of interacting elements, such as electronic circuits, social communication, metabolism or gene regulation. Network inference, analysis, and modeling techniques have been developed in different areas of science and technology, such as computer science, mathematics, physics, and biology, with an active interdisciplinary exchange of concepts and approaches. However, some concepts seem to belong to a specific field without a clear transferability to other domains. At the same time, it is increasingly recognized that within some biological systems-such as the tumor microenvironment-where different types of resident and infiltrating cells interact to carry out their functions, the complexity of the system demands a theoretical framework, such as statistical inference, graph analysis and dynamical models, in order to asses and study the information derived from high-throughput experimental technologies. RESULTS: In this article we propose to adopt and adapt the concepts of influence and investment from the world of social network analysis to biological problems, and in particular to apply this approach to infer causality in the tumor microenvironment. We showed that constructing a bidirectional network of influence between cell and cell communication molecules allowed us to determine the direction of inferred regulations at the expression level and correctly recapitulate cause-effect relationships described in literature. CONCLUSIONS: This work constitutes an example of a transfer of knowledge and concepts from the world of social network analysis to biomedical research, in particular to infer network causality in biological networks. This causality elucidation is essential to model the homeostatic response of biological systems to internal and external factors, such as environmental conditions, pathogens or treatments

TIE-2 expressing monocytes in human cancers.

Tumor-associated macrophages (TAM) are well known as a key player in the tumor microenvironment, which support cancer progression. More recently, a lineage of monocytes characterized by the expression of the TIE-2/Tek angiopoietin receptor identified a subset of circulating and tumor-associated monocytes endowed with proangiogenic activity. TIE-2 expressing monocytes (TEM) were found both in humans and mice. Here, we review the phenotypes and functions of TEM reported so far in human cancer and their potential use as markers of cancer progression and metastasis. Finally, we discuss the therapeutic approaches currently used or proposed to target TEM

Identifying biological mechanisms for favorable cancer prognosis using non-hypothesis-driven iterative survival analysis.

Survival analyses based on the Kaplan-Meier estimate have been pervasively used to support or validate the relevance of biological mechanisms in cancer research. Recently, with the appearance of gene expression high-throughput technologies, this kind of analysis has been applied to tumor transcriptomics data. In a 'bottom-up' approach, gene-expression profiles that are associated with a deregulated pathway hypothetically involved in cancer progression are first identified and then subsequently correlated with a survival effect, which statistically supports or requires the rejection of such a hypothesis. In this work, we propose a 'top-down' approach, in which the clinical outcome (survival) is the starting point that guides the identification of deregulated biological mechanisms in cancer by a non-hypothesis-driven iterative survival analysis. We show that the application of our novel method to a population of ~2,000 breast cancer patients of the METABRIC consortium allows the identification of several well-known cancer mechanisms, such as ERBB4, HNF3A and TGFB pathways, and the investigation of their paradoxical dual effect. In addition, several novel biological mechanisms are proposed as potentially involved in cancer progression. The proposed exploratory methodology can be considered both alternative and complementary to classical 'bottom-up' approaches for validation of biological hypotheses. We propose that our method may be used to better characterize cancer, and may therefore impact the future design of therapies that are truly molecularly tailored to individual patients. The method, named SURCOMED, was implemented as a web-based tool, which is publicly available at http://surcomed.vital-it.ch. R scripts are also available at http://surcomed.sourceforge.net)

Noninvasive rapid detection of metabolic adaptation in activated human T lymphocytes by hyperpolarized ¹³C magnetic resonance.

The metabolic shift induced in human CD4 <sup>+</sup> T lymphocytes by stimulation is characterized by an upregulation of glycolysis, leading to an augmentation in lactate production. This adaptation has already been highlighted with various techniques and reported in several previous studies. We herein propose a method to rapidly and noninvasively detect the associated increase in flux from pyruvate to lactate catalyzed by lactate dehydrogenase using hyperpolarized <sup>13</sup> C magnetic resonance, a technique which can be used for in vivo imaging. It was shown that the conversion of hyperpolarized <sup>13</sup> C-pyruvate to <sup>13</sup> C-lactate during the one-minute measurement increased by a mean factor of 3.6 in T cells stimulated for 5 days as compared to resting T cells. This method can be extended to other metabolic substrates and is therefore a powerful tool to noninvasively analyze T cell metabolism, possibly in vivo

Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer.

Bone marrow-derived endothelial progenitor cells (EPCs) infiltrate into sites of neovascularization in adult tissues and mature into functional blood endothelial cells (BECs) during a process called vasculogenesis. Human marrow-derived EPCs have recently been reported to display a mixed myeloid and lymphatic endothelial cell (LEC) phenotype during inflammation-induced angiogenesis; however, their role in cancer remains poorly understood. We report the in vitro differentiation of human cord blood CD133(+)CD34(+) progenitors into podoplanin(+) cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers vascular endothelium growth factor receptor 3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and prospero homeobox 1 (PROX-1). These podoplanin(+) cells displayed sprouting behavior comparable to that of LECs in vitro and a dual hemangiogenic and lymphangiogenic activity in vivo in an endothelial cell sprouting assay and corneal vascularization assay, respectively. Furthermore, these cells expressed vascular endothelium growth factor (VEGF) family members A, -C, and -D. Thus, bone-marrow derived EPCs stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34(+) cord blood progenitors into hemangiogenic and lymphangiogenic CD11b(+) myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b(+) cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer

Fine-Tuning of Optimal TCR Signaling in Tumor-Redirected CD8 T Cells by Distinct TCR Affinity-Mediated Mechanisms

Redirecting CD8 T cell immunity with self/tumor-specific affinity-matured T cell receptors (TCRs) is a promising approach for clinical adoptive T cell therapy, with the aim to improve treatment efficacy. Despite numerous functional-based studies, little is known about the characteristics of TCR signaling (i.e., intensity, duration, and amplification) and the regulatory mechanisms underlying optimal therapeutic T cell responses. Using a panel of human SUP-T1 and primary CD8 T cells engineered with incremental affinity TCRs against the cancer-testis antigen NY-ESO-1, we found that upon activation, T cells with optimal-affinity TCRs generated intense and sustained proximal (CD3 zeta, LCK) signals associated with distal (ERK1/2) amplification-gain and increased function. In contrast, in T cells with very high affinity TCRs, signal initiation was rapid and strong yet only transient, resulting in poor MAPK activation and low proliferation potential even at high antigen stimulation dose. Under resting conditions, the levels of surface TCR/CD3e, CD8 beta, and CD28 expression and of CD3. phosphorylation were significantly reduced in those hypo-responsive cells, suggesting the presence of TCR affinity-related activation thresholds. We also show that SHP phosphatases were involved along the TCR affinity gradient, but displayed spatially distinct regulatory roles. While PTPN6/SHP-1 phosphatase activity controlled TCR signaling initiation and subsequent amplification by counteracting CD3. and ERK1/2 phosphorylation, PTPN11/SHP-2 augmented MAPK activation without affecting proximal TCR signaling. Together, our findings indicate that optimal TCR signaling can be finely tuned by TCR affinity-dependent SHP-1 and SHP-2 activity, and this may readily be determined at the TCR/CD3 complex level. We propose that these TCR affinity-associated regulations represent potential protective mechanisms preventing high affinity TCR-mediated autoimmune diseases

TIE-2-expressing monocytes are lymphangiogenic and associate specifically with lymphatics of human breast cancer.

In experimental mouse models of cancer, increasingly compelling evidence point toward a contribution of tumor associated macrophages (TAM) to tumor lymphangiogenesis. Corresponding experimental observations in human cancer remain scarce although lymphatic metastasis is widely recognized as a predominant route for tumor spread. We previously showed that, in malignant tumors of untreated breast cancer (BC) patients, TIE-2-expressing monocytes (TEM) are highly proangiogenic immunosuppressive cells and that TIE-2 and VEGFR signaling pathways drive TEM immunosuppressive function. We report here that, in human BC, TEM express the canonical lymphatic markers LYVE-1, Podoplanin, VEGFR-3 and PROX-1. Critically, both TEM acquisition of lymphatic markers and insertion into lymphatic vessels were observed in tumors but not in adjacent non-neoplastic tissues, suggesting that the tumor microenvironment shapes both TEM phenotype and spatial distribution. We assessed the lymphangiogenic activity of TEM isolated from dissociated primary breast tumors in vitro and in vivo using endothelial cells (EC) sprouting assay and corneal vascularization assay, respectively. We show that, in addition to their known hemangiogenic function, TEM isolated from breast tumor display a lymphangiogenic activity. Importantly, TIE-2 and VEGFR pathways display variable contributions to TEM angiogenic and lymphangiogenic activities across BC patients; however, combination of TIE-2 and VEGFR kinase inhibitors abrogated these activities and overcame inter-patient variability. These results highlight the direct contribution of tumor TEM to the breast tumor lymphatic network and suggest a combined use of TIE-2 and VEGFR kinase inhibitors as a therapeutic approach to block hem- and lymphangiogenesis in BC

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rβ-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential

Nanowire Sensors in Cancer.

In 2006, the group of Dr C.M. Lieber pioneered the field of nanowire sensors by fabricating devices for the ultra-sensitive label-free detection of biological macromolecules. Since then, nanowire sensors have demonstrated their ability to detect cancer-associated analytes in peripheral blood, tumor tissue, and the exhaled breath of cancer patients. These innovative developments have marked a new era with unprecedented detection performance, capable of addressing crucial needs such as cancer diagnosis and monitoring disease progression and patient response to therapy. The ability of nanowire sensors to identify molecular features of patient tumor represents a first step toward precision medicine, and their integration into portable devices has the potential to revolutionize cancer diagnosis and patient monitoring