Innate lymphoid cells and tumor-derived lactic acid: novel contenders in an enduring game.

Aerobic glycolysis, also known as the Warburg effect, has for a prolonged period of time been perceived as a defining feature of tumor metabolism. The redirection of glucose utilization towards increased production of lactate by cancer cells enables their rapid proliferation, unceasing growth, and longevity. At the same time, it serves as a significant contributor to acidification of the tumor microenvironment, which, in turn, imposes substantial constraints on infiltrating immune cells. Here, we delve into the influence of tumor-derived lactic acid on innate lymphoid cells (ILCs) and discuss potential therapeutic approaches. Given the abundance of ILCs in barrier tissues such as the skin, we provide insights aimed at translating this knowledge into therapies that may specifically target skin cancer.

Cancer immunosurveillance by ILC2s.

Group 2 innate lymphoid cells (ILC2s) elicit ostensibly paradoxical responses, such as tissue repair and stimulation of tumorigenesis. Given emerging evidence that ILC2s also contribute to cancer immunosurveillance, we reassess the role of ILC2s in tumorigenesis and discuss recent insights into their tumoricidal potential.

Pharmacokinetics and Pharmacodynamics of T-Cell Bispecifics in the Tumour Interstitial Fluid.

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies.

Innate Lymphoid Cells in Skin Homeostasis and Malignancy.

Innate lymphoid cells (ILCs) are mostly tissue resident lymphocytes that are preferentially enriched in barrier tissues such as the skin. Although they lack the expression of somatically rearranged antigen receptors present on T and B cells, ILCs partake in multiple immune pathways by regulating tissue inflammation and potentiating adaptive immunity. Emerging evidence indicates that ILCs play a critical role in the control of melanoma, a type of skin malignancy thought to trigger immunity mediated mainly by adaptive immune responses. Here, we compile our current understanding of ILCs with regard to their role as the first line of defence against melanoma development and progression. We also discuss areas that merit further investigation. We envisage that the possibility to harness therapeutic potential of ILCs might benefit patients suffering from skin malignancies such as melanoma.

Tumor-Derived Lactic Acid Contributes to the Paucity of Intratumoral ILC2s.

Group 2 innate lymphoid cells (ILC2s) are abundant in non-lymphoid tissues and increase following infectious and inflammatory insults. In solid tumors, however, ILC2s constitute a relatively small proportion of immune cells. Here, we show, using melanoma as a model, that while the IL-33/IL C2/eosinophil axis suppresses tumor growth, tumor-derived lactate attenuates the function and survival of ILC2s. Melanomas with reduced lactate production (LDHAlow) are growth delayed and typified by an increased number of ILC2s compared with control tumors. Upon IL-33 stimulation, ILC2s accompanied by eosinophils more effectively restrain the growth of LDHAlow tumors than control melanomas. Furthermore, database analysis reveals a negative correlation between the expression of LDHA and markers associated with ILC2s and the association of high expression of IL33 and an eosinophil marker SIGLEC8 with better overall survival in human cutaneous melanoma patients. This work demonstrates that the balance between the IL-33/ILC2/eosinophil axis and lactate production by tumor cells regulates melanoma growth.

A 3D Skin Melanoma Spheroid-Based Model to Assess Tumor-Immune Cell Interactions.

Three-dimensional (3D) tumor spheroids have the potential to bridge the gap between two-dimensional (2D) monolayer tumor cell cultures and solid tumors with which they share a significant degree of similarity. However, the progression of solid tumors is often influenced by the dynamic and reciprocal interactions between tumor and immune cells. Here we present a 3D tumor spheroid-based model that might shed new light on understanding the mechanisms of tumor and immune cell interactions. The model first utilizes the hanging drop assay, which serves as one of the simplest methods for generating 3D spheroids and requires no specialized equipment. Next, pre-established spheroids can be co-cultured either directly or indirectly with an immune cell population of interest. Using skin melanoma, we provide a detailed description of the model, which might hold a significant importance for the development of successful therapeutic strategies.

Blockade of Lymphangiogenesis Shapes Tumor-Promoting Adipose Tissue Inflammation.

Tumor-associated lymphangiogenesis correlates with lymph node metastasis and poor outcome in several human malignancies. In addition, the presence of functional lymphatic vessels regulates the formation of tumor inflammatory and immune microenvironments. Although lymphatic structures are often found deeply integrated into the fabric of adipose tissue, the impact of lymphangiogenesis on tumor-associated adipose tissue (AT) has not yet been investigated. Using K14-VEGFR3-Ig mice that constitutively express soluble vascular endothelial growth factor receptor (VEGFR) 3-Ig in the skin, scavenging VEGF-C and VEGF-D, the role of lymphangiogenesis in the generation of an inflammatory response within tumor-associated AT was studied. Macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 were found within peritumoral adipose tissue from melanoma-bearing K14-VEGFR3-Ig mice, which were further enriched with alternatively activated macrophages based on surface marker CD301/C-type lectin domain family 10 member A expression. The blockade of lymphangiogenesis also resulted in accumulation of the cytokine IL-6, which correlated with enhanced macrophage proliferation of the alternatively activated phenotype. Furthermore, melanomas co-implanted with freshly isolated adipose tissue macrophages grew more robustly than melanomas growing alone. In human cutaneous melanomas, adipocyte-selective FABP4 transcripts closely correlated with gene signatures of CLEC10A and were associated with poor overall survival. These data suggest that the blockade of pathways regulating lymphatic vessel formation shapes an inflammatory response within tumor-associated AT by facilitating accumulation of tumor-promoting alternatively activated macrophages.

Cancer Immunoediting by Innate Lymphoid Cells.

The immune system plays a dual role in cancer. It conveys protective immunity but also facilitates malignant progression, either by sculpting tumor immunogenicity or by creating a microenvironment that can stimulate tumor outgrowth or aid in a subsequent metastatic cascade. Innate lymphoid cells (ILCs) embody this functional heterogeneity, although the nature of their responses in cancer has only recently begun to be unveiled. We provide an overview of recent insights into the role of ILCs in cancer. We also discuss how ILCs fit into the conceptual framework of cancer immunoediting, which integrates the dual role of the immune system in carcinogenesis. A broader understanding of their relevance in cancer is essential towards the design of successful therapeutic strategies.

Hide and seek: Plasticity of innate lymphoid cells in cancer.

The advance of immunotherapies has revolutionized the treatment of cancer patients. Mostly agents modulating the adaptive immune system are currently used. More recently, attempts to stimulate the innate immune system are being promoted for clinical evaluation. Innate lymphoid cells (ILCs) are a highly plastic population of immune cells crucial for tissue homeostasis and the regulation of immune responses and maybe a promising target to improve current cancer immunotherapies. Although we have made significant progress in understanding ILC biology, their impact on tumor development, progression and therapy is controversial. In this review, we discuss the recent advances of ILC function and plasticity in the context of cancer.

Plastic Heterogeneity of Innate Lymphoid Cells in Cancer.

Innate lymphoid cells (ILCs) fulfill important protective and reparative functions, and have thus been implicated in the maintenance of tissue homeostasis. Dysregulation of their activation is associated with several autoimmune and inflammatory diseases. The current literature on the role of ILCs in cancer is limited and our knowledge is therefore incomplete. Indeed, ILCs have been separately associated with tumor-promoting as well as tumor-suppressing activities, raising the need to understand the mechanisms by which these cells regulate tumor growth and progression toward a rational design of therapeutic approaches. We focus here on the heterogeneity of ILCs and discuss currently known mechanisms of their plasticity.

Lymphatic vessels regulate immune microenvironments in human and murine melanoma.

Lymphatic remodeling in tumor microenvironments correlates with progression and metastasis, and local lymphatic vessels play complex and poorly understood roles in tumor immunity. Tumor lymphangiogenesis is associated with increased immune suppression, yet lymphatic vessels are required for fluid drainage and immune cell trafficking to lymph nodes, where adaptive immune responses are mounted. Here, we examined the contribution of lymphatic drainage to tumor inflammation and immunity using a mouse model that lacks dermal lymphatic vessels (K14-VEGFR3-Ig mice). Melanomas implanted in these mice grew robustly, but exhibited drastically reduced cytokine expression and leukocyte infiltration compared with those implanted in control animals. In the absence of local immune suppression, transferred cytotoxic T cells more effectively controlled tumors in K14-VEGFR3-Ig mice than in control mice. Furthermore, gene expression analysis of human melanoma samples revealed that patient immune parameters are markedly stratified by levels of lymphatic markers. This work suggests that the establishment of tumor-associated inflammation and immunity critically depends on lymphatic vessel remodeling and drainage. Moreover, these results have implications for immunotherapies, the efficacies of which are regulated by the tumor immune microenvironment.

Impaired lymphatic function accelerates cancer growth.

Increased lymphangiogenesis is a common feature of cancer development and progression, yet the influence of impaired lymphangiogenesis on tumor growth is elusive. C3HBA breast cancer and KHT-1 sarcoma cell lines were implanted orthotopically in Chy mice, harboring a heterozygous inactivating mutation of vascular endothelial growth factor receptor-3, resulting in impaired dermal lymphangiogenesis. Accelerated tumor growth was observed in both cancer models in Chy mice, coinciding with reduced peritumoral lymphangiogenesis. An impaired lymphatic washout was observed from the peritumoral area in Chy mice with C3HBA tumors, and the number of macrophages was significantly reduced. While fewer macrophages were detected, the fraction of CD163+ M2 macrophages remained constant, causing a shift towards a higher M2/M1 ratio in Chy mice. No difference in adaptive immune cells was observed between wt and Chy mice. Interestingly, levels of pro- and anti-inflammatory macrophage-associated cytokines were reduced in C3HBA tumors, pointing to an impaired innate immune response. However, IL-6 was profoundly elevated in the C3HBA tumor interstitial fluid, and treatment with the anti-IL-6 receptor antibody tocilizumab inhibited breast cancer growth. Collectively, our data indicate that impaired lymphangiogenesis weakens anti-tumor immunity and favors tumor growth at an early stage of cancer development.

Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.

Tumor Interstitial Fluid Formation, Characterization, and Clinical Implications.

The interstitium, situated between the blood and lymph vessels and the cells, consists of a solid or matrix phase and a fluid phase representing the tissue microenvironment. In the present review, we focus on the interstitial fluid phase of solid tumors, the tumor interstitial fluid (TIF), i.e., the fluid bathing the tumor and stroma cells, also including immune cells. This is a component of the internal milieu of a solid tumor that has attracted regained attention. Access to this space may provide important insight into tumor development and therapy response. TIF is formed by transcapillary filtration, and since this fluid is not readily available we discuss available techniques for TIF isolation, results from subsequent characterization and implications of recent findings with respect to fluid filtration and uptake of macromolecular therapeutic agents. There appear to be local gradients in signaling substances from neoplastic tissue to plasma that may provide new understanding of tumor biology. The development of sensitive proteomic technologies has made TIF a valuable source for tumor specific proteins and biomarker candidates. Potential biomarkers will appear locally in high concentrations in tumors and may eventually be found diluted in the plasma. Access to TIF that reliably reflects the local tumor microenvironment enables identification of substances that can be used in early detection and monitoring of disease.

Adipose tissue macrophages: the inflammatory link between obesity and cancer?

INTRODUCTION: Obesity has increased dramatically over the last three decades. Thus, epidemiological evidence linking obesity and cancer has ignited our interest in the relationship between adipose tissue mass and cancer development. Obesity is defined as an excess of adipose tissue that is typified by a chronic, low-grade inflammatory response instigated by macrophage infiltration. Therefore, in this review, we will discuss the putative causal relationship between obesity-induced chronic inflammation and cancer with particular focus on adipose tissue macrophages. AREAS COVERED: Chronic, low-grade inflammation has long been associated with cancer initiation, promotion and progression. Therefore, signals derived from adipose tissue macrophages may play a significant role in carcinogenesis. In this review we will discuss the molecular mechanisms of cancer development in obesity and highlight possible therapeutic strategies aiming at adipose tissue macrophages. EXPERT OPINION: The strong correlation between tumor-associated macrophage infiltration and tumor growth and progression emphasizes the value of macrophages as an effective therapeutic target. It remains to be deciphered to what extent adipose tissue macrophages contribute to these processes, especially in tumors growing within or adjacent to adipose tissue. More effort should also be placed on elucidating macrophage differences between humans and mice that may lead to the development of more effective diagnostic and therapeutic strategies.

A dangerous duo in adipose tissue: high-mobility group box 1 protein and macrophages.

High-mobility group box 1 (HMGB1) protein first made headlines 40 years ago as a non-histone nuclear protein that regulates gene expression. Not so long ago, it was also shown that HMGB1 has an additional surprising function. When released into the extracellular milieu, HMGB1 triggers an inflammatory response by serving as an endogenous danger signal. The pro-inflammatory role of HMGB1 is now well-established and has been associated with several diseases, including sepsis, rheumatoid arthritis, and atherosclerosis. Yet very little is known about its role in obesity, wherein adipose tissue is typified by a persistent, smoldering inflammatory response instigated by high macrophage infiltrate that potentiates the risk of obesity-associated comorbidities. This mini-review focuses on the putative causal relationship between HMGB1 and macrophage pro-inflammatory activation in pathologically altered adipose tissue associated with obesity.

Tumor versus stromal cells in culture--survival of the fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.

Loss of adipocyte specification and necrosis augment tumor-associated inflammation.

Most tumors are typified by a chronic, unresolved inflammatory response that potentiates angiogenesis and therefore enables tumor progression. We have determined that dysfunctional tumor-associated adipocytes contribute to tumor-associated inflammation. In three tumor models, tumor-associated adipose tissue was characterized by thin and fragile adipocyte membranes, necrosis, robust expression of the pro-inflammatory factor HMGB1, and loss of the lipid storage mediator, perilipin-1. By transmission electron microscopy, macrophages in tumor-associated adipose tissue contained lipid droplets and resembled foam cells, which are commonly observed in inflamed tissues. In vitro co-culture studies showed that tumor-associated adipose tissue conditioned-medium stimulated monocyte-to-macrophage differentiation, adhesion, spreading, and lipid uptake. Compared with normal adipose tissue, tumor-associated adipose tissue secreted 3-fold higher levels of IL-6 and IL-6 was sufficient to stimulate macrophage differentiation and adhesion. These results suggest that, in tumors, loss of adipocyte specification, necrosis, and scavenging of adipocyte debris directly activates macrophages and contributes to tumor-associated inflammation. Thus, adipocyte dysfunction may facilitate tumor progression, especially in tumors closely aligned with adipose tissue, in particular, breast cancer.

A three-party alliance in solid tumors: Adipocytes, macrophages and vascular endothelial cells.

In tumors, cross talk between malignant and non-malignant cells (stroma) influences tumor growth, angiogenesis and metastasis. Stromal cells in tumors typically include vascular cells, fibroblasts and a heterogeneous population of inflammatory cells. Adipocytes may also be present. Adipose tissue is perhaps the least studied stromal cell "compartment" despite the fact that some tumors, particularly breast tumors, grow in close proximity to or physically interact with adipocytes. Apart from adipocytes and numerous blood vessels, adipose tissue harbors macrophages, which increase in proportion to adipose tissue mass. While circulating or bone marrow-derived macrophages play a well-defined role in tumor growth, it is less understood how resident adipose tissue-associated macrophages contribute to tumor progression. Here, we will review the role of adipose tissue in tumor growth and angiogenesis with emphasis on the specific functions of adipose tissue macrophages in these processes.

Increased interstitial protein because of impaired lymph drainage does not induce fibrosis and inflammation in lymphedema.

OBJECTIVE: The pathophysiology of lymphedema is incompletely understood. We asked how transcapillary fluid balance parameters and lymph flow are affected in a transgenic mouse model of primary lymphedema, which due to an inhibition of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling lacks dermal lymphatics, and whether protein accumulation in the interstitium occurring in lymphedema results in inflammation. METHODS AND RESULTS: As estimated using a new optical-imaging technique, we found that this signaling defect resulted in lymph drainage in hind limb skin of K14-VEGFR-3-Ig mice that was 34% of the corresponding value in wild-type. The interstitial fluid pressure and tissue fluid volumes were significantly increased in the areas of visible swelling only, whereas the colloid osmotic pressure in plasma, and thus the colloid osmotic pressure gradient, was reduced compared to wild-type mice. An acute volume load resulted in an exaggerated interstitial fluid pressure response in transgenic mice. There was no accumulation of collagen or lipid in skin, suggesting that chronic edema presented in the K14-VEGFR-3-Ig mouse was not sufficient to induce changes in tissue composition. Proinflammatory cytokines (interleukin-2, interleukin-6, interleukin-12) in subcutaneous interstitial fluid and macrophage infiltration in skin of the paw were lower, whereas the monocyte/macrophage cell fraction in blood and spleen was higher in transgenic compared with wild-type mice. CONCLUSIONS: Our data suggest that a high interstitial protein concentration and longstanding edema is not sufficient to induce fibrosis and inflammation characteristic for the human condition and may have implications for our understanding of the pathophysiology of this condition.