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Supplementary Figure from Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer

The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte–macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.

Supplementary Table from Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer

PDF file 401K, List of p38MAPK-dependent SASP factors and their overlap with publically available expression analyses from breast cancer-associated microenvironments

PDF file 62K, legends for supplemental figures

PDF file 229K, qRT-PCR validation of RNA-seq analysis and TNFalpha ELISA on p38i treated mice

PDF file 545K, SASP upregulation following bleomycin, NaB or replication-induced senescence. SASP mRNA stabilization in NaB-treated BJs and in bleomycin-treated IMR90s

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PDF file 194K, Senescence-associated beta-galactosidase staining of BJ fibroblasts and treatment of HaCaT-CBR cells with the p38MAPK small-molecule inhibitor SB203580

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The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte–macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.

The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T cell function and their relation to patient outcome. Here we leverage single-cell RNA-sequencing (scRNA-seq) analysis of several mouse and human tumors and find that monocyte-macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. Using mouse models, we also find that tumor monocyte-to-macrophage progression is profoundly tied to regulatory T cell (Treg) abundance. Importantly, in human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states in kidney tumors.

Matthew Krummel and colleagues discuss the positive trends and initiatives that emerged among scientists during the COVID-19 lockdown., The response to the COVID-19 crisis across most research institutions mandated ceasing nonessential research activities in order to minimize the spread of the virus in our communities. With minimal notice, experiments were terminated, cell lines were frozen, mouse colonies were culled, and trainees were prevented from performing bench research. Still, despite the interruption of experimental productivity, the shutdown has proven for many PIs and trainees that doing and thinking science are not activities that are bound to the laboratory. Furthermore, the shutdowns have solidified important emerging trends and forced us to further innovate to get the most out of working remotely. We hope that some of these innovations, hard-gained in this difficult time, will persist and develop into new paradigms—lessons that will improve our science and our relationship to the climate and community beyond the current pandemic.

Tumors frequently program CD8 T cell immunity that is ‘exhausted’, or otherwise fails to provide for tumor clearance. This begins when tumor antigens reach the lymph node (LN) to stimulate T cells yet we know little of how tumor material is transported and disseminated amongst the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, in vitro and in vivo assays together with lattice light sheet and multiphoton imaging we demonstrate that tumor proteins are carried to LN within discrete vesicles inside DC and are then transferred amongst DC subsets. A cup-on-cup synapse is formed between interacting DC and directed vesicle transfer takes place routinely in the absence of free exosomes. DC containing these vesicles can uniquely activate T cells whereas DC lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.

In order to drive productive tumor-infiltrating lymphocyte (TIL) function, myeloid populations must direct antigens to the lymph node, including to resident antigen-presenting cells (APCs) that have never touched the tumor. It has long been supposed that APCs trade antigens with one another, but the dominant cell biology underlying that remains unknown. We used in vitro and in vivo assays together with lattice light sheet and multiphoton imaging to show that myeloid cells carry tumor antigen-laden vesicles that they ‘trade’ with one another as they reach distant sites. This accounts for the majority of antigen displayed to T cells and provides tumors with a mechanism to access APCs that differentially direct T cell activation away from memory phenotypes. This work defines efficient cell biology that drives the first steps of TIL generation and represents a new frontier for engineering tumoral immunity.

Differentiation of proinflammatory CD4(+) conventional T cells (T(conv)) are critical for productive antitumor responses yet their elicitation remains poorly understood. We exhaustively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4(+) T(conv), but then fail to support antitumor CD4(+) T(conv) differentiation. Regulatory T cell (T(reg)) depletion enhanced their capacity to elicit strong CD4(+) T(conv) responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice their abundance relative to T(reg) predicts protective ICOS(+) PD-1(lo) CD4(+) T(conv) phenotypes and survival. Further, in melanoma patients with low T(reg) abundance, intratumoral cDC2 density alone correlates with abundant CD4(+) T(conv) and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway which restrains cDC2, and whose reversal enhances CD4(+) T(conv) abundance and controls tumor growth.

SummaryMore than 85% of advanced breast cancer patients suffer from metastatic bone lesions, yet the mechanisms that facilitate these metastases remain poorly understood. Recent studies suggest that tumor-derived factors initiate changes within the tumor microenvironment to facilitate metastasis. However, whether stromal-initiated changes are sufficient to drive increased metastasis in the bone remains an open question. Thus, we developed a model to induce reactive senescent osteoblasts and found that they increased breast cancer colonization of the bone. Analysis of senescent osteoblasts revealed that they failed to mineralize bone matrix and increased local osteoclastogenesis, the latter process being driven by the senescence-associated secretory phenotype factor, IL-6. Neutralization of IL-6 was sufficient to limit senescence-induced osteoclastogenesis and tumor cell localization to bone, thereby reducing tumor burden. Together, these data suggest that a reactive stromal compartment can condition the niche, in the absence of tumor-derived signals, to facilitate metastatic tumor growth in the bone.

The immune system must maintain a delicate balance between activation and tolerance. It is required to quickly respond and eliminate a variety of threats while at the same time remain unresponsive upon encountering normal self-cells or commensal bacteria. We have created a system to track antigen trafficking throughout the immune system and using this system have identified a pathway of antigen flow from peripheral tissues and tumors to the draining lymph node (LN). By tracking antigen from steady-state tissues and tumors, we find that antigen is handled differentially in the LN depending on the inflammatory context in the periphery. During pronounced inflammation, self-antigen shows altered trafficking consistent with that of tumor antigen. Interestingly, using both gut and skin systems, commensal bacteria-derived antigen displayed the unique quality of cell type specific uptake. While each of the various migratory dendritic cell (DC) types were proficient at uptake of self and tumor antigens, only migratory CD11b+ DC and macrophages showed detectable commensal antigen uptake. Additionally, tracking of commensal bacteria-derived antigen within DC showed minimal antigen trafficking to the LN, suggesting different modes of maintaining immune tolerance are at play depending on the antigen source. We find that self-antigen can drain to the LN via migratory DC but is limited from passing to resident DC populations, which appears to prevent robust self-reactive T-cell priming. However, in the case of commensal antigen, the restriction of antigen flow is early in the pathway, thus effectively keeping the LN ignorant of antigen. Identifying the signals that restrict antigen flow in cases of immune tolerance will likely provide insight into how tumor antigen availability affects immune activation. Understanding how self, commensal and tumor antigens are handled by immune cells under various contexts will help inform the development of new targeted immunotherapies that seek to activate or inhibit immune activation in cases of cancer and autoimmunity. Citation Format: Megan K. Ruhland, Edward W. Roberts, Matthew F. Krummel. Modulating antigen flow to control immune tolerance [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B184.

Neoplastic cells rely on the tumor microenvironment (TME) for survival and progression factors. Indeed, senescent and cancer-associated fibroblasts (CAF) express factors that promote tumorigenesis that are collectively referred to as the senescence-associated secretory phenotype (SASP). Despite their importance in tumorigenesis, the mechanisms that control TME-derived factor expression remain poorly understood. Here, we address a key unanswered question: how the SASP is sustained in senescent fibroblasts and CAFs. We find that the mitogen-activated protein kinase p38 (p38MAPK) controls AUF1 occupancy on SASP mRNAs and thus controls their stability. The importance of this regulatory mechanism is underscored by our findings that stromal-specific p38MAPK inhibition abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Our data suggest that targeting SASP mRNA stability through inhibition of p38MAPK will significantly aid the development of clinical strategies to target the TME. Significance: The TME plays a key role in tumorigenesis. We demonstrate that p38MAPK governs a posttranscriptional mechanism that sustains the protumorigenic SASP. Inhibition of p38MAPK abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Thus, p38MAPK is a TME-specific Achilles' heel that may be exploited as a new therapeutic target. Cancer Discov; 4(6); 716–29. ©2014 AACR. See related commentary by Isacke and Barcellos-Hoff, p. 637 This article is highlighted in the In This Issue feature, p. 621

Alterations in the microenvironment collaborate with cell autonomous mutations during the transformation process. Indeed, cancer-associated fibroblasts and senescent fibroblasts stimulate tumorigenesis in xenograft models. Because senescent fibroblasts accumulate with age, these findings suggest that they contribute to age-related increases in tumorigenesis. Previously we showed that senescence-associated stromal-derived osteopontin contributes to preneoplastic cell growth in vitro and in xenografts, suggesting that it impacts neoplastic progression. Analysis of fibroblasts within premalignant and malignant skin lesions ranging from solar/actinic keratosis to squamous cell carcinoma revealed they express osteopontin. Given the stromal expression of osteopontin, we investigated how osteopontin impacts preneoplastic cell growth. We show that osteopontin promotes preneoplastic keratinocyte cellular proliferation and cell survival through the CD44 cell receptor and activation of the MAPK pathway. These data suggest that stromal-derived osteopontin impacts tumorigenesis by stimulating preneoplastic cell proliferation thus allowing expansion of initiated cells in early lesions. Mol Cancer Res; 9(8); 1018–29. ©2011 AACR.

The immune response to tumors is malleable as we have learned from the various success of immunotherapies across a diversity of tumor types. What are the foundations for a solid immune response, particularly one executed by T cells? Imaging and single-cell approaches provide key insights into immune cell types that can be unleashed on tumors, identifying both their identity and the immune interactions that are to be fostered. We discuss a few of these with particular emphasis on the diversity of the myeloid system and how its component parts can contribute toward antitumor immunity. Citation Format: Adriana Mujal, Kevin Barry, Edward Roberts, Mikhail Binnewies, Megan Ruhland, Vincent Chan, Matthew Frederick Krummel. Imaging- and single cell- based insights into the antitumor immune responses [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA05.

The senescent phenotype was first described in 1961 as a phenomenon characterized by the cessation of cellular division. After years of debate as to whether it represented a tissue culture artifact or an important biological process, it is now appreciated that senescence plays an important role in tumorigenesis. Further, senescence is integral to normal biological processes such as embryogenesis and the maintenance of tissue homeostasis. Now with defined roles in development, wound healing, tumor promotion and tumor suppression, it is not surprising that attention has turned to refining our understanding of the mechanisms behind, and consequences of, the induction of senescence. One emerging role for senescence lies in the ability of senescence to orchestrate an inflammatory response: factors secreted by senescent cells have been identified in multiple contexts to modulate various aspects of the immune response. As with many of the previously described roles for senescence, the type of inflammation established by the senescence phenotype is varied and dependent on context. In this review, we discuss the current state of the field with a focus on the paradoxical outcomes of the senescence-induced inflammatory responses in the context of cancer. A more complete understanding of senescence and an appreciation for its complexities will be important for eventual development of senescence-targeted therapies.