Turning “Cold” Into “Hot” Tumors—Opportunities and Challenges for Radio-Immunotherapy Against Primary and Metastatic Brain Cancers

The development of immunotherapies has revolutionized intervention strategies for a variety of primary cancers. Despite this promising progress, treatment options for primary brain cancer and brain metastasis remain limited and still largely depend on surgical resection, radio- and/or chemotherapy. The paucity in the successful development of immunotherapies for brain cancers can in part be attributed to the traditional view of the brain as an immunologically privileged site. The presence of the blood-brain barrier and the absence of lymphatic drainage were believed to restrict the entry of blood-borne immune and inflammatory cells into the central nervous system (CNS), leading to an exclusion of the brain from systemic immune surveillance. However, recent insight from pre-clinical and clinical studies on the immune landscape of brain cancers challenged this dogma. Recruitment of blood-borne immune cells into the CNS provides unprecedented opportunities for the development of tumor microenvironment (TME)-targeted or immunotherapies against primary and metastatic cancers. Moreover, it is increasingly recognized that in addition to genotoxic effects, ionizing radiation represents a critical modulator of tumor-associated inflammation and synergizes with immunotherapies in adjuvant settings. This review summarizes current knowledge on the cellular and molecular identity of tumor-associated immune cells in primary and metastatic brain cancers and discusses underlying mechanisms by which ionizing radiation modulates the immune response. Detailed mechanistic insight into the effects of radiation on the unique immune landscape of brain cancers is essential for the development of multimodality intervention strategies in which immune-modulatory effects of radiotherapy are exploited to sensitize brain cancers to immunotherapies by converting immunologically “cold” into “hot” environments

Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death

Brain-Resident Microglia and Blood-Borne Macrophages Orchestrate Central Nervous System Inflammation in Neurodegenerative Disorders and Brain Cancer

Inflammation is a hallmark of different central nervous system (CNS) pathologies. It has been linked to neurodegenerative disorders as well as primary and metastatic brain tumors. Microglia, the brain-resident immune cells, are emerging as a central player in regulating key pathways in CNS inflammation. Recent insights into neuroinflammation indicate that blood-borne immune cells represent an additional critical cellular component in mediating CNS inflammation. The lack of experimental systems that allow for discrimination between brain-resident and recruited myeloid cells has previously halted functional analysis of microglia and their blood-borne counterparts in brain malignancies. However, recent conceptual and technological advances, such as the generation of lineage tracing models and the identification of cell type-specific markers provide unprecedented opportunities to study the cellular functions of microglia and macrophages by functional interference. The use of different “omic” strategies as well as imaging techniques has significantly increased our knowledge of disease-associated gene signatures and effector functions under pathological conditions. In this review, recent developments in evaluating functions of brain-resident and recruited myeloid cells in neurodegenerative disorders and brain cancers will be discussed and unique or shared cellular traits of microglia and macrophages in different CNS disorders will be highlighted. Insight from these studies will shape our understanding of disease- and cell-type-specific effector functions of microglia or macrophages and will open new avenues for therapeutic intervention that target aberrant functions of myeloid cells in CNS pathologies

TAMs in brain metastasis: molecular signatures in mouse and man

Macrophages not only represent an integral part of innate immunity but also critically contribute to tissue and organ homeostasis. Moreover, disease progression is accompanied by macrophage accumulation in many cancer types and is often associated with poor prognosis and therapy resistance. Given their critical role in modulating tumor immunity in primary and metastatic brain cancers, macrophages are emerging as promising therapeutic targets. Different types of macrophages infiltrate brain cancers, including (i) CNS resident macrophages that comprise microglia (TAM-MG) as well as border-associated macrophages and (ii) monocyte-derived macrophages (TAM-MDM) that are recruited from the periphery. Controversy remained about their disease-associated functions since classical approaches did not reliably distinguish between macrophage subpopulations. Recent conceptual and technological advances, such as large-scale omic approaches, provided new insight into molecular profiles of TAMs based on their cellular origin. In this review, we summarize insight from recent studies highlighting similarities and differences of TAM-MG and TAM-MDM at the molecular level. We will focus on data obtained from RNA sequencing and mass cytometry approaches. Together, this knowledge significantly contributes to our understanding of transcriptional and translational programs that define disease-associated TAM functions. Cross-species meta-analyses will further help to evaluate the translational significance of preclinical findings as part of the effort to identify candidates for macrophage-targeted therapy against brain metastasis

Human cathepsin L rescues the neurodegeneration and lethality in cathepsin B/L double deficient mice

Cathepsin B (CTSB) and cathepsin L (CTSL) are two widely expressed cysteine proteases thought to predominantly reside within lysosomes. Functional analysis of CTSL in humans is complicated by the existence of two CTSL-like homologues (CTSL and CTSL2), in contrast to mice which contain only one CTSL enzyme. Thus transgenic expression of human CTSL in CTSL deficient mice provides an opportunity to study the in vivo functions of this human protease without interference by its highly related homologue. While mice with single gene deficiencies for murine CTSB or CTSL survive without apparent neuromuscular impairment, murine CTSB/CTSL double deficient mice display degeneration of cerebellar Purkinje cells and neurons of the cerebral cortex, resulting in severe hypotrophy, motility defects, and lethality during their third to fourth week of life. Here we show that expression of human CTSL through a genomic transgene results in widespread expression of human CTSL in the mouse which is capable of rescuing the lethality found in CTSB/CTSL double-deficient animals. Human CTSL is expressed in the brain of these compound mutants predominantly in neurons of the cerebral cortex and in Purkinje cells of the cerebellum, where it appears to prevent neuronal cell death

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern

Microenvironmental regulation of tumor progression and therapeutic response in brain metastasis

Cellular and non-cellular components of the tumor microenvironment (TME) are emerging as key regulators of primary tumor progression, organ-specific metastasis, and therapeutic response. In the era of TME-targeted- and immunotherapies, cancer-associated inflammation has gained increasing attention. In this regard, the brain represents a unique and highly specialized organ. It has long been regarded as an immunological sanctuary site where the presence of the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCB) restricts the entry of immune cells from the periphery. Consequently, tumor cells that metastasize to the brain were thought to be shielded from systemic immune surveillance and destruction. However, the detailed characterization of the immune landscape within border-associated areas of the central nervous system (CNS), such as the meninges and the choroid plexus, as well as the discovery of lymphatics and channels that connect the CNS with the periphery, have recently challenged the dogma of the immune privileged status of the brain. Moreover, the presence of brain metastases (BrM) disrupts the integrity of the BBB and BCB. Indeed, BrM induce the recruitment of different immune cells from the myeloid and lymphoid lineage to the CNS. Blood-borne immune cells together with brain-resident cell-types, such as astrocytes, microglia, and neurons, form a highly complex and dynamic TME that affects tumor cell survival and modulates the mode of immune responses that are elicited by brain metastatic tumor cells. In this review, we will summarize recent findings on heterotypic interactions within the brain metastatic TME and highlight specific functions of brain-resident and recruited cells at different rate-limiting steps of the metastatic cascade. Based on the insight from recent studies, we will discuss new opportunities and challenges for TME-targeted and immunotherapies for BrM

Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity

Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes

The Brain Pre-Metastatic Niche: Biological and Technical Advancements

Metastasis, particularly brain metastasis, continues to puzzle researchers to this day, and exploring its molecular basis promises to break ground in developing new strategies for combatting this deadly cancer. In recent years, the research focus has shifted toward the earliest steps in the formation of metastasis. In this regard, significant progress has been achieved in understanding how the primary tumor affects distant organ sites before the arrival of tumor cells. The term pre-metastatic niche was introduced for this concept and encompasses all influences on sites of future metastases, ranging from immunological modulation and ECM remodeling to the softening of the blood–brain barrier. The mechanisms governing the spread of metastasis to the brain remain elusive. However, we begin to understand these processes by looking at the earliest steps in the formation of metastasis. This review aims to present recent findings on the brain pre-metastatic niche and to discuss existing and emerging methods to further explore the field. We begin by giving an overview of the pre-metastatic and metastatic niches in general before focusing on their manifestations in the brain. To conclude, we reflect on the methods usually employed in this field of research and discuss novel approaches in imaging and sequencing