Enabling research into the tumour microenvironment: Novel Photonics Assays for Cancer Research

Physics and engineering principles have long been applied to the development of instrumentation and assays that have significantly advanced biological research. In particular, photonics based instruments and assays have proven to be powerful tools that enable researchers to investigate biological processes in vivo. The research described in this thesis covers a range of photonics based instruments and assays that expand the capabilities researchers have to investigate challenging biological problems. These advances give researchers new tools for directly visualising dynamic biological events in three ways: 1) How to look: novel microscope instrumentation; 2) What to look at: Novel imaging based assays to dissect the tumour microenvironment; and 3) Where to look: Novel surgical protocols to enable ultra-high-resolution optical imaging in living animals (intravital imaging). Application of these assays and instruments to the challenging problem of cancer metastasis has led to a new understanding of the process of intravasation and haematogenous dissemination, paving the way to new clinical diagnostics and therapies

Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible β-Actin – Dendra2 Fusion Proteins

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin - Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells

A unidirectional transition from migratory to perivascular macrophage is required for tumor cell intravasation

Summary: Tumor-associated macrophages (TAMs) are critical for tumor metastasis. Two TAM subsets support cancer cell intravasation: migratory macrophages guide cancer cells toward blood vessels, where sessile perivascular macrophages assist their entry into the blood. However, little is known about the inter-relationship between these functionally distinct TAMs or their possible inter-conversion. We show that motile, streaming TAMs are newly arrived monocytes, recruited via CCR2 signaling, that then differentiate into the sessile perivascular macrophages. This unidirectional process is regulated by CXCL12 and CXCR4. Cancer cells induce TGF-β-dependent upregulation of CXCR4 in monocytes, while CXCL12 expressed by perivascular fibroblasts attracts these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiate into perivascular macrophages, promoting vascular leakiness and intravasation. : Tumor-associated macrophages (TAMs) are essential for metastasis. Arwert et al. show that, following extravasation, monocytes initially become motile TAMs. Tumor-derived TGF-β then induces CXCR4 on TAMs, stimulating them to migrate toward CXCL12-expressing perivascular fibroblasts. Once adjacent to blood vessels, TAMs differentiate into metastasis-assisting perivascular TAMs. Keywords: tumor associated macrophages, TAMs, TGF beta, breast cancer, metastasis, CXCR4, CCR2, TMEM, Men

Interleukin 4 Controls the Pro-Tumoral Role of Macrophages in Mammary Cancer Pulmonary Metastasis in Mice

SIMPLE SUMMARY: Metastasis is the main cause of death from breast cancer. In mouse models of breast cancer lung metastasis, macrophages enhance metastasis by promoting tumor cell seeding and persistent growth. Here, we show that interleukin-4 (IL4) is required for this process as IL4 receptor (IL4rα)-null mice develop fewer and smaller lung metastases. This deficiency is partially rescued by adoptive transfer of wild-type monocytes. IL4 signaling in macrophages upregulates the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. In addition, expression of several other genes already causally associated with lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1 are upregulated in macrophages. High-resolution intravital imaging at the time of metastatic seeding showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages, showing the dependence on IL4. We conclude that IL4 signaling in monocytes and macrophages is important during seeding and growth of breast metastasis in the lung. ABSTRACT: Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected transendothelial migration of tumor cells in vitro. Interferon gamma and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4rα)-null mice developed fewer and smaller lung metastasis in E0771-LG mammary cancer models of this disease. Adoptive transfer of wild-type monocytes to IL4rα-deficient mice partially rescued this phenotype. IL4 signaling in macrophages controlled the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages regulated the transcript abundance of several other genes already causally associated with mammary cancer lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1. The central role of IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages. This interaction with wild-type MAMs enhanced tumor cell survival and seeding, which was lost in the IL4rα mice. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung, as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages

A metastasis biomarker (MetaSite Breast™ Score) is associated with distant recurrence in hormone receptor-positive, HER2-negative early-stage breast cancer

Metastasis is the primary cause of death in early-stage breast cancer. We evaluated the association between a metastasis biomarker, which we call "Tumor Microenviroment of Metastasis" (TMEM), and risk of recurrence. TMEM are microanatomic structures where invasive tumor cells are in direct contact with endothelial cells and macrophages, and which serve as intravasation sites for tumor cells into the circulation. We evaluated primary tumors from 600 patients with Stage I-III breast cancer treated with adjuvant chemotherapy in trial E2197 (NCT00003519), plus endocrine therapy for hormone receptor (HR)+ disease. TMEM were identified and enumerated using an analytically validated, fully automated digital pathology/image analysis method (MetaSite Breast™), hereafter referred to as MetaSite Score (MS). The objectives were to determine the association between MS and distant relapse free interval (DRFI) and relapse free interval (RFI). MS was not associated with tumor size or nodal status, and correlated poorly with Oncotype DX Recurrence Score (r = 0.29) in 297 patients with HR+/HER2- disease. Proportional hazards models revealed a significant positive association between continuous MS and DRFI (p = 0.001) and RFI (p = 0.00006) in HR+/HER2- disease in years 0-5, and by MS tertiles for DRFI (p = 0.04) and RFI (p = 0.01), but not after year 5 or in triple negative or HER2+ disease. Multivariate models in HR+/HER- disease including continuous MS, clinical covariates, and categorical Recurrence Score ( 30) showed MS is an independent predictor for 5-year RFI (p = 0.05). MetaSite Score provides prognostic information for early recurrence complementary to clinicopathologic features and Recurrence Score.Breast Cancer Research Foundatio

Breast Cancer Cell Re-Dissemination from Lung Metastases—A Mechanism for Enhancing Metastatic Burden

Although metastatic disease is the primary cause of mortality in cancer patients, the mechanisms leading to overwhelming metastatic burden are still incompletely understood. Metastases are the endpoint of a series of multi-step events involving cancer cell intravasation, dissemination to distant organs, and outgrowth to metastatic colonies. Here we show, for the first-time, that breast cancer cells do not solely disseminate to distant organs from primary tumors and metastatic nodules in the lymph nodes, but also do so from lung metastases. Thus, our findings indicate that metastatic dissemination could continue even after the removal of the primary tumor. Provided that the re-disseminated cancer cells initiate growth upon arrival to distant sites, cancer cell re-dissemination from metastatic foci could be one of the crucial mechanisms leading to overt metastases and patient demise. Therefore, the development of new therapeutic strategies to block cancer cell re-dissemination would be crucial to improving survival of patients with metastatic disease

Macrophages Promote Tumor Cell Extravasation across an Endothelial Barrier through Thin Membranous Connections

Macrophages are important players involved in the progression of breast cancer, including in seeding the metastatic niche. However, the mechanism by which macrophages in the lung parenchyma interact with tumor cells in the vasculature to promote tumor cell extravasation at metastatic sites is not clear. To mimic macrophage-driven tumor cell extravasation, we used an in vitro assay (eTEM) in which an endothelial monolayer and a matrigel-coated filter separated tumor cells and macrophages from each other. The presence of macrophages promoted tumor cell extravasation, while macrophage conditioned media was insufficient to stimulate tumor cell extravasation in vitro. This finding is consistent with a requirement for direct contact between macrophages and tumor cells. We observed the presence of Thin Membranous Connections (TMCs) resembling similar structures formed between macrophages and tumor cells called tunneling nanotubes, which we previously demonstrated to be important in tumor cell invasion in vitro and in vivo. To determine if TMCs are important for tumor cell extravasation, we used macrophages with reduced levels of endogenous M-Sec (TNFAIP2), which causes a defect in tunneling nanotube formation. As predicted, these macrophages showed reduced macrophage-tumor cell TMCs. In both, human and murine breast cancer cell lines, there was also a concomitant reduction in tumor cell extravasation in vitro when co-cultured with M-Sec deficient macrophages compared to control macrophages. We also detected TMCs formed between macrophages and tumor cells through the endothelial layer in the eTEM assay. Furthermore, tumor cells were more frequently found in pores under the endothelium that contain macrophage protrusions. To determine the role of macrophage-tumor cell TMCs in vivo, we generated an M-Sec deficient mouse. Using an in vivo model of experimental metastasis, we detected a significant reduction in the number of metastatic lesions in M-Sec deficient mice compared to wild type mice. There was no difference in the size of the metastases, consistent with a defect specific to tumor cell extravasation and not metastatic outgrowth. Additionally, with an examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lungs, we could detect the presence of TMCs between extravascular macrophages and vascular tumor cells. Overall, our data indicate that macrophage TMCs play an important role in promoting the extravasation of circulating tumor cells in the lungs