T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control.

Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet the mechanisms of lymphocyte transit are not well defined. Here we show that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression by effector CD8+ T cells. Only high-affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells, therefore, exit the tumor, which limits the pool of CD8+ T cells available to exert tumor control. CXCR4 inhibition or loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. These data indicate that strategies to limit T cell egress might be an approach to boost the quantity and quality of intratumoral T cells and thereby response to immunotherapy.

The Basis of Oncoimmunology.

Cancer heterogeneity, a hallmark enabling clonal survival and therapy resistance, is shaped by active immune responses. Antigen-specific T cells can control cancer, as revealed clinically by immunotherapeutics such as adoptive T-cell transfer and checkpoint blockade. The host immune system is thus a powerful tool that, if better harnessed, could significantly enhance the efficacy of cytotoxic therapy and improve outcomes for cancer sufferers. To realize this vision, however, a number of research frontiers must be tackled. These include developing strategies for neutralizing tumor-promoting inflammation, broadening T-cell repertoires (via vaccination), and elucidating the mechanisms by which immune cells organize tumor microenvironments to regulate T-cell activity. Such efforts will pave the way for identifying new targets for combination therapies that overcome resistance to current treatments and promote long-term cancer control.

Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy.

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging.

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.

Metabolic abnormalities in the bone marrow cells of young offspring born to mothers with obesity.

BACKGROUND/OBJECTIVES: Intrauterine metabolic reprogramming occurs in mothers with obesity during gestation, putting the offspring at high risk of developing obesity and associated metabolic disorders even before birth. We have generated a mouse model of maternal high-fat diet-induced obesity that recapitulates the metabolic changes seen in humans born to women with obesity. METHODS: Here, we profiled and compared the metabolic characteristics of bone marrow cells of newly weaned 3-week-old offspring of dams fed either a high-fat (Off-HFD) or a regular diet (Off-RD). We utilized a state-of-the-art flow cytometry, and targeted metabolomics approach coupled with a Seahorse metabolic analyzer. RESULTS: We revealed significant metabolic perturbation in the offspring of HFD-fed vs. RD-fed dams, including utilization of glucose primarily via oxidative phosphorylation. We also show a reduction in levels of amino acids, a phenomenon previously linked to bone marrow aging. Using flow cytometry, we found changes in the immune complexity of bone marrow cells and identified a unique B cell population expressing CD19 and CD11b in the bone marrow of three-week-old offspring of high-fat diet-fed mothers. Our data also revealed increased expression of Cyclooxygenase-2 (COX-2) on myeloid CD11b, and on CD11bhi B cells. CONCLUSIONS: Altogether, we demonstrate that the offspring of mothers with obesity show metabolic and immune changes in the bone marrow at a very young age and prior to any symptomatic metabolic disease.

An open protocol for modeling T Cell Clonotype repertoires using TCRß CDR3 sequences.

T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.

Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

Some DCs are "B"etter.

In this issue of Immunity, Ma et al. (2013) demonstrate that neoplastic cell release of ATP drives recruitment and differentiation of CD11b(+) dendritic cells within tumors, where their ability to locally present antigen improves response to anthracycline-based chemotherapy.

High IRF8 expression correlates with CD8 T cell infiltration and is a predictive biomarker of therapy response in ER-negative breast cancer.

BACKGROUND: Characterization of breast cancer (BC) through the determination of conventional markers such as ER, PR, HER2, and Ki67 has been useful as a predictive and therapeutic tool. Also, assessment of tumor-infiltrating lymphocytes has been proposed as an important prognostic aspect to be considered in certain BC subtypes. However, there is still a need to identify additional biomarkers that could add precision in distinguishing therapeutic response of individual patients. To this end, we focused in the expression of interferon regulatory factor 8 (IRF8) in BC cells. IRF8 is a transcription factor which plays a well-determined role in myeloid cells and that seems to have multiple antitumoral roles: it has tumor suppressor functions; it acts downstream IFN/STAT1, required for the success of some therapeutic regimes, and its expression in neoplastic cells seems to depend on a cross talk between the immune contexture and the tumor cells. The goal of the present study was to examine the relationship between IRF8 with the therapeutic response and the immune contexture in BC, since its clinical significance in this type of cancer has not been thoroughly addressed. METHODS: We identified the relationship between IRF8 expression and the clinical outcome of BC patients and validated IRF8 as predictive biomarker by using public databases and then performed in silico analysis. To correlate the expression of IRF8 with the immune infiltrate in BC samples, we performed quantitative multiplex immunohistochemistry. RESULTS: IRF8 expression can precisely predict the complete pathological response to monoclonal antibody therapy or to select combinations of chemotherapy such as FAC (fluorouracil, adriamycin, and cytoxan) in ER-negative BC subtypes. Analysis of immune cell infiltration indicates there is a strong correlation between activated and effector CD8+ T cell infiltration and tumoral IRF8 expression. CONCLUSIONS: We propose IRF8 expression as a potent biomarker not only for prognosis, but also for predicting therapy response in ER-negative BC phenotypes. Its expression in neoplastic cells also correlates with CD8+ T cell activation and infiltration. Therefore, our results justify new efforts towards understanding mechanisms regulating IRF8 expression and how they can be therapeutically manipulated.

Cathepsin C is a tissue-specific regulator of squamous carcinogenesis.

Serine and cysteine cathepsin (Cts) proteases are an important class of intracellular and pericellular enzymes mediating multiple aspects of tumor development. Emblematic of these is CtsB, reported to play functionally significant roles during pancreatic islet and mammary carcinogenesis. CtsC, on the other hand, while up-regulated during pancreatic islet carcinogenesis, lacks functional significance in mediating neoplastic progression in that organ. Given that protein expression and enzymatic activity of both CtsB and CtsC are increased in numerous tumors, we sought to understand how tissue specificity might factor into their functional significance. Thus, whereas others have reported that CtsB regulates metastasis of mammary carcinomas, we found that development of squamous carcinomas occurs independently of CtsB. In contrast to these findings, our studies found no significant role for CtsC during mammary carcinogenesis but revealed squamous carcinogenesis to be functionally dependent on CtsC. In this context, dermal/stromal fibroblasts and bone marrow-derived cells expressed increased levels of enzymatically active CtsC that regulated the complexity of infiltrating immune cells in neoplastic skin, development of angiogenic vasculature, and overt squamous cell carcinoma growth. These studies highlight the important contribution of tissue/microenvironment context to solid tumor development and indicate that tissue specificity defines functional significance for these two members of the cysteine protease family.

Robust Cell Detection and Segmentation for Image Cytometry Reveal Th17 Cell Heterogeneity.

Image cytometry enables quantitative cell characterization with preserved tissue architecture; thus, it has been highlighted in the advancement of multiplex immunohistochemistry (IHC) and digital image analysis in the context of immune-based biomarker monitoring associated with cancer immunotherapy. However, one of the challenges in the current image cytometry methodology is a technical limitation in the segmentation of nuclei and cellular components particularly in heterogeneously stained cancer tissue images. To improve the detection and specificity of single-cell segmentation in hematoxylin-stained images (which can be utilized for recently reported 12-biomarker chromogenic sequential multiplex IHC), we adapted a segmentation algorithm previously developed for hematoxlin and eosin-stained images, where morphological features are extracted based on Gabor-filtering, followed by stacking of image pixels into n-dimensional feature space and unsupervised clustering of individual pixels. Our proposed method showed improved sensitivity and specificity in comparison with standard segmentation methods. Replacing previously proposed methods with our method in multiplex IHC/image cytometry analysis, we observed higher detection of cell lineages including relatively rare TH 17 cells, further enabling sub-population analysis into TH 1-like and TH 2-like phenotypes based on T-bet and GATA3 expression. Interestingly, predominance of TH 2-like TH 17 cells was associated with human papilloma virus (HPV)-negative status of oropharyngeal squamous cell carcinoma of head and neck, known as a poor-prognostic subtype in comparison with HPV-positive status. Furthermore, TH 2-like TH 17 cells in HPV-negative head and neck cancer tissues were spatiotemporally correlated with CD66b+ granulocytes, presumably associated with an immunosuppressive microenvironment. Our cell segmentation method for multiplex IHC/image cytometry potentially contributes to in-depth immune profiling and spatial association, leading to further tissue-based biomarker exploration. © 2019 International Society for Advancement of Cytometry.

Immune microenvironments in solid tumors: new targets for therapy.

Leukocytes and their soluble mediators play important regulatory roles in all aspects of solid tumor development. While immunotherapeutic strategies have conceptually held clinical promise, with the exception of a small percentage of patients, they have failed to demonstrate effective, consistent, and durable anti-cancer responses. Several subtypes of leukocytes that commonly infiltrate solid tumors harbor immunosuppressive activity and undoubtedly restrict the effectiveness of these strategies. Several of these same immune cells also foster tumor development by expression of potent protumor mediators. Given recent evidence revealing that immune-based mechanisms regulate the response to conventional cytotoxic therapy, it seems reasonable to speculate that tumor progression could be effectively diminished by combining cytotoxic strategies with therapies that blunt protumor immune-based effectors and/or neutralize those that instead impede development of desired anti-tumor immunity, thus providing synergistic effects between traditional cytotoxic and immune-modulatory approaches.

Senescence and cancer: An evolving inflammatory paradox.

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.

A Listeria vaccine and depletion of T-regulatory cells activate immunity against early stage pancreatic intraepithelial neoplasms and prolong survival of mice.

BACKGROUND & AIMS: Premalignant lesions and early stage tumors contain immunosuppressive microenvironments that create barriers for cancer vaccines. Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which express an activated form of Kras in pancreatic tissues, develop pancreatic intraepithelial neoplasms (PanIN) that progress to pancreatic ductal adenocarcinoma (PDA). We used these mice to study immune suppression in PDA. METHODS: We immunized KPC and Kras(G12D/+);Pdx-1-Cre mice with attenuated intracellular Listeria monocytogenes (which induces CD4(+) and CD8(+) T-cell immunity) engineered to express Kras(G12D) (LM-Kras). The vaccine was given alone or in sequence with an anti-CD25 antibody (PC61) and cyclophosphamide to deplete T-regulatory (Treg) cells. Survival times were measured; pancreatic and spleen tissues were collected and analyzed by histologic, flow cytometry, and immunohistochemical analyses. RESULTS: Interferon γ-mediated, CD8(+) T-cell responses were observed in KPC and Kras(G12D/+);Pdx-1-Cre mice given LM-Kras, but not in unvaccinated mice. Administration of LM-Kras to KPC mice 4-6 weeks old (with early stage PanINs), depleted of Treg cells, significantly prolonged survival and reduced PanIN progression (median survival, 265 days), compared with unvaccinated mice (median survival, 150 days; P = .002), mice given only LM-Kras (median survival, 150 days; P = .050), and unvaccinated mice depleted of Treg cells (median survival, 170 days; P = .048). In 8- to 12-week-old mice (with late-stage PanINs), LM-Kras, alone or in combination with Treg cell depletion, did not increase survival time or slow PanIN progression. The combination of LM-Kras and Treg cell depletion reduced numbers of Foxp3(+)CD4(+) T cells in pancreatic lymph nodes, increased numbers of CD4(+) T cells that secrete interleukin 17 and interferon γ, and caused CD11b(+)Gr1(+) cells in the pancreas to acquire an immunostimulatory phenotype. CONCLUSIONS: Immunization of KPC mice with Listeria monocytogenes engineered to express Kras(G12D), along with depletion of Treg cells, reduces progression of early stage, but not late-stage, PanINs. This approach increases infiltration of the lesion with inflammatory cells. It might be possible to design immunotherapies against premalignant pancreatic lesions to slow or prevent progression to PDA.

Differential macrophage programming in the tumor microenvironment.

Of the multiple unique stromal cell types common to solid tumors, tumor-associated macrophages (TAMs) are significant for fostering tumor progression. The protumor properties of TAMs derive from regulation of angiogenic programming, production of soluble mediators that support proliferation, survival and invasion of malignant cells, and direct and indirect suppression of cytotoxic T cell activity. These varied activities are dependent on the polarization state of TAMs that is regulated in part by local concentrations of cytokines and chemokines, as well as varied interactions of TAMs with normal and degraded components of the extracellular matrix. Targeting molecular pathways regulating TAM polarization holds great promise for anticancer therapy.

Tumor-infiltrating regulatory T cells inhibit endogenous cytotoxic T cell responses to lung adenocarcinoma.

Immune cells comprise a substantial proportion of the tumor mass in human nonsmall cell lung cancers (NSCLC), but the precise composition and significance of this infiltration are unclear. In this study, we examined immune complexity of human NSCLC as well as NSCLC developing in CC10-TAg transgenic mice, and revealed that CD4(+) T lymphocytes represent the dominant population of CD45(+) immune cells, and, relative to normal lung tissue, CD4(+)Foxp3(+) regulatory T cells (Tregs) were significantly increased as a proportion of total CD4(+) cells. To assess the functional significance of increased Tregs, we evaluated CD8(+) T cell-deficient/CC10-TAg mice and revealed that CD8(+) T cells significantly controlled tumor growth with antitumor activity that was partially repressed by Tregs. However, whereas treatment with anti-CD25-depleting mAb as monotherapy preferentially depleted Tregs and improved CD8(+) T cell-mediated control of tumor progression during early tumor development, similar monotherapy was ineffective at later stages. Because mice bearing early NSCLC treated with anti-CD25 mAb exhibited increased tumor cell death associated with infiltration by CD8(+) T cells expressing elevated levels of granzyme A, granzyme B, perforin, and IFN-γ, we therefore evaluated carboplatin combination therapy resulting in a significantly extended survival beyond that observed with chemotherapy alone, indicating that Treg depletion in combination with cytotoxic therapy may be beneficial as a treatment strategy for advanced NSCLC.

Paradoxical roles of the immune system during cancer development.

The main function of the mammalian immune system is to monitor tissue homeostasis, to protect against invading or infectious pathogens and to eliminate damaged cells. Therefore, it is surprising that cancer occurs with such a high frequency in humans. Recent insights that have been gained from clinical studies and experimental mouse models of carcinogenesis expand our understanding of the complex relationship between immune cells and developing tumours. Here, we examine the paradoxical role of adaptive and innate leukocytes as crucial regulators of cancer development and highlight recent insights that have been gained by manipulating immune responses in mouse models of de novo and spontaneous tumorigenesis.

Leukocyte composition of human breast cancer.

Retrospective clinical studies have used immune-based biomarkers, alone or in combination, to predict survival outcomes for women with breast cancer (BC); however, the limitations inherent to immunohistochemical analyses prevent comprehensive descriptions of leukocytic infiltrates, as well as evaluation of the functional state of leukocytes in BC stroma. To more fully evaluate this complexity, and to gain insight into immune responses after chemotherapy (CTX), we prospectively evaluated tumor and nonadjacent normal breast tissue from women with BC, who either had or had not received neoadjuvant CTX before surgery. Tissues were evaluated by polychromatic flow cytometry in combination with confocal immunofluorescence and immunohistochemical analysis of tissue sections. These studies revealed that activated T lymphocytes predominate in tumor tissue, whereas myeloid lineage cells are more prominant in "normal" breast tissue. Notably, residual tumors from an unselected group of BC patients treated with neoadjuvant CTX contained increased percentages of infiltrating myeloid cells, accompanied by an increased CD8/CD4 T-cell ratio and higher numbers of granzyme B-expressing cells, compared with tumors removed from patients treated primarily by surgery alone. These data provide an initial evaluation of differences in the immune microenvironment of BC compared with nonadjacent normal tissue and reveal the degree to which CTX may alter the complexity and presence of selective subsets of immune cells in tumors previously treated in the neoadjuvant setting.