Tumor-infiltrating Immune Populations Research Articles

Sequence of androgen receptor-targeted vaccination with androgen deprivation therapy affects anti-prostate tumor efficacy

RationaleAndrogen deprivation therapy (ADT) is the primary treatment for recurrent and metastatic prostate cancer. In addition to direct antitumor effects, ADT has immunomodulatory effects such as promoting T-cell infiltration and...

Abstract 1179: A single-cell atlas of human gastric malignancy reveals CD8+ T cell rejuvenation as a mediator of immunotherapeutic response

Abstract Immune checkpoint inhibition (ICI) has emerged as a mainstream treatment for patients with advanced stage gastric cancer (GC). Cellular heterogeneity in the tumor microenvironment (TME) has a profound impact on the therapeutic responses to ICI. Here we characterize the kinetics of tumor-infiltrating immune populations in GC patients treated with anti-PD-1 using single-cell RNA and TCR sequencing (scRNA/TCR-seq), including primary tumors and metastases. Integrated with public GC scRNA-seq datasets, we construct the largest multi-omic atlas of GC cell states to date that captures the full trajectory of GC malignancy and comprises >320,000 cells derived from 73 samples. We demonstrate the rejuvenation of exhausted CD8+ T cells into a proliferative state marked by the loss of LAYN expression as a major mediator of ICI response in the TME. In parallel, we observe enhanced immunogenicity of cancer cells in post-ICI tumors, thus highlighting the coordination between tumor-intrinsic and microenvironmental factors elicited by ICI. Our study provides a valuable resource of investigating the tumor response to anti-PD-1 treatment and suggests novel strategies for combination therapy and patient stratification. Citation Format: Han Liang, Jian Chen, Yikai Luo, Muxing Kang, Wei Liu, Lie Wang. A single-cell atlas of human gastric malignancy reveals CD8+ T cell rejuvenation as a mediator of immunotherapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1179.

Abstract 5169: Aurora-A kinase promotes anti-tumor immunity in high lymphocyte-infiltrated colorectal cancer

Abstract Introduction: The tumor microenvironment (TME) is composed of different cell types and is involved in tumor progression. Several immunosuppressive signaling pathways can promote tumor growth by inhibiting anti-tumor immunity in TEM. Therefore, targeting the immunosuppressive signaling pathways is a potential cancer treatment. Recent studies reported that inhibition of oncoproteins could lead to reduced anti-tumor immunity in several cancers; therefore, it’s critical to identify the potential oncoproteins which can lead to enhanced anti-tumor immunity. Aurora-A is a serine/threonine kinase that plays an important role in the centrosome, cell mitosis, and chromosome stability. Previous studies indicated that cancer patients with poor prognoses are associated with a high Aurora-A level in tumor cells. Inhibition of Aurora-A is considered as a promising strategy for cancer treatment. However, Aurora-A inhibitors have not had a breakthrough in phase II or phase III clinical trials. The role of Aurora-A in tumor immunity is still unclear. Here, we investigated the role of Aurora-A in tumor immunity. Materials and Methods: shRNA was used to knock down Aurora-A, and Alisertib was added to inhibit Aurora-A. RT-qPCR and western blot were performed to determine the IL-16 expression. The secretion of cytokines was measured by cytokine array and ELISA. BALB/c and NOD/SCID mice were subcutaneously injected with CT26 and then administered daily with Alisertib or treated with anti-IL-16 antibody. The tumor-infiltrating immune populations were analyzed by flow cytometry. IHC and H/E stain were performed to analyze Aurora-A expression and lymphocyte infiltration in human CRC specimens. Results: CRC patients with lower lymphocyte infiltration and higher Aurora-A are associated with poor prognosis and vice versa. Knockdown of Aurora-A in CT26 promotes tumor growth in immunocompetent mice by inhibiting the infiltration and cytotoxic activity of CD8+ T cells. Aurora-A negatively regulates IL-16 expression in a kinase-dependent manner in CRC cells. Blockade of IL-16 by anti-IL-16 antibody can reverse the tumor growth and CD8+ T cell cytotoxic activity in Aurora-A knocked-down CT26 tumors. Alisertib can inhibit CT26 tumor growth and promote IL-16 secretion in TME in mice. A combination of Alisertib and IL-16 antibody can promote the therapeutic effect of Alisertib in CT26 tumors. Conclusion: Tumor-intrinsic Aurora-A plays a diverse role in tumor progression depending on the status of lymphocyte infiltration in TME. Aurora-A can promote anti-tumor immunity via negatively regulating IL-16 in a kinase activity-dependent manner in hot CRC tumors. In tumors with high lymphocyte infiltration, Aurora-A inhibition can suppress cancer cell growth and reduce anti-tumor immunity; the combination of Aurora-A inhibitors and IL-16 antibodies may provide a novel and effective strategy for cancer therapy in hot tumors. Citation Format: Shiang-Jie Yang, Ming-Derg Lai, Liang-Yi Hung. Aurora-A kinase promotes anti-tumor immunity in high lymphocyte-infiltrated colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5169.

Abstract 6665: A spatiotemporal cell atlas of human gastric malignancy reveals mechanisms underlying metastasis and immunotherapy response

Abstract Cellular heterogeneity in the tumor immune microenvironment (TIME) of gastric cancer (GC) is progressively molded in pre- and early-malignant lesions and has a profound impact on disease progression and responses to therapeutics, in particular those based on immune checkpoint blockade (ICB). To date, no study has been conducted to characterize the single-cell kinetics of tumor-infiltrating immune populations in gastric cancer patients treated with ICB. Here, we generated for the first time single-cell RNA sequencing (scRNA-seq) and TCR sequencing (scTCR-seq) profiles of ICB-treated primary GC tumors, as well as liver and ovary metastases. When integrated with public scRNA-seq datasets, our data capture a full trajectory of GC malignancy comprising more than 300,000 cells derived from 73 samples. We further performed bulk TCR-seq, cytometry by time of flight (CyTOF), and multiplex immunofluorescence (mxIF) on independent GC samples to orthogonally validate the key observations from the scRNA-seq cohort and unbiasedly search for more in-depth and spatial-relationship-focused features of ICB-responsive immune populations. Built upon this multi-omic atlas of GC cell states, we identified remarkable transformation of the abundance and clonal dynamics of distinct immune populations and their population-specific transcriptional programs. Most importantly, we observed the rejuvenation of exhausted CD8+ T cells induced by anti-PD-1 treatment, which is associated with the enhanced immunogenicity of gastric cancer cells. In addition, we found the enrichment of NK-like terminally differentiated CD8+ T cells in metastases independent of migration destinations. Our single-cell ICB-perturbed spatiotemporal GC landscape reveals how neoadjuvant checkpoint blockade induces local and systemic tumor immunity. As a cellular and molecular reference, it also enables high-resolution interpretation of future single-cell omics data in gastric cancer that may be masked by de novo analysis. Citation Format: Yikai Luo, Muxing Kang, Wei Liu, Shiping Jiao, Lie Wang, Jian Chen, Han Liang. A spatiotemporal cell atlas of human gastric malignancy reveals mechanisms underlying metastasis and immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6665.

CFI-402257, a TTK inhibitor, effectively suppresses hepatocellular carcinoma

Deregulation of cell cycle is a typical feature of cancer cells. Normal cells rely on the strictly coordinated spindle assembly checkpoint (SAC) to maintain the genome integrity and survive. However, cancer cells could bypass this checkpoint mechanism. In this study, we showed the clinical relevance of threonine tyrosine kinase (TTK) protein kinase, a central regulator of the SAC, in hepatocellular carcinoma (HCC) and its potential as therapeutic target. Here, we reported that a newly developed, orally active small molecule inhibitor targeting TTK (CFI-402257) effectively suppressed HCC growth and induced highly aneuploid HCC cells, DNA damage, and micronuclei formation. We identified that CFI-402257 also induced cytosolic DNA, senescence-like response, and activated DDX41-STING cytosolic DNA sensing pathway to produce senescence-associated secretory phenotypes (SASPs) in HCC cells. These SASPs subsequently led to recruitment of different subsets of immune cells (natural killer cells, CD4+ T cells, and CD8+ T cells) for tumor clearance. Our mass cytometry data illustrated the dynamic changes in the tumor-infiltrating immune populations after treatment with CFI-402257. Further, CFI-402257 improved survival in HCC-bearing mice treated with anti-PD-1, suggesting the possibility of combination treatment with immune checkpoint inhibitors in HCC patients. In summary, our study characterized CFI-402257 as a potential therapeutic for HCC, both used as a single agent and in combination therapy.

IMMU-11. SELECTIVE ENRICHMENT OF SUPPRESSED IMMUNE CELLS IN THE TUMOR MICROENVIRONMENT CORRELATES WITH ESTABLISHMENT OF DISTINCT IMMUNOLOGIC NICHES IN HUMAN GLIOBLASTOMAS CONTACTING THE LATERAL VENTRICLE

Abstract Glioblastomas (GBM) account for 60% of adult primary brain tumors. With few advances in therapeutics, median overall survival remains 15-months post-diagnosis. Immunotherapies may provide therapeutic benefit in GBM patients; however, no predictive immune features currently inform therapeutic stratification in GBM. We have shown that, independently of known prognosticators, radiographic tumor contact with the lateral ventricle (C-GBM) correlates with 7-months worse prognosis compared to patients with ventricle non-contacting GBM (NC-GBM). This study sought to characterize the GBM immune microenvironment and identify targetable mechanisms of immunosuppression correlating with worse outcomes in C-GBM. Primary glioblastoma specimens were resected in accordance with the Declaration of Helsinki (IRB #131870). Twelve patients presented with C-GBM and thirteen with NC-GBM. Machine learning tools applied to mass cytometry data characterized tumor-infiltrating immune populations and identified biomarkers correlating with C-GBM and patient survival. C-GBM tumors were enriched in blood-derived macrophages compared to NC-GBM (19 ± 8% vs. 6 ± 2%; p< 0.001) and depleted in lymphocytes (2.9 ± 1% vs. 7.6 ± 2%; p< 0.001) and tissue-resident microglia (1.8 ± 0.3% vs. 7 ± 3%; p< 0.001). Further, T cells in C-GBM co-expressed the checkpoint receptors PD-1 and TIGIT, suggesting acute T cell exhaustion. Multiplex immunohistochemistry (mxIHC) on matched FFPE tissue provided spatial context to risk-stratifying immune populations, and defined structured immunological niches within the TME. Macrophage-tumor niches were most common (36%), followed by T cell-microglia-tumor niches (26%). Within niches, T cell-T cell interactions were more prevalent (log odds ratio = 0.90) whereas T cell-macrophage interactions were less prevalent (log odds ratio = -1.61). These findings suggest that factors within the periventricular space may influence antitumor immunity within tumors, and identify clinically targetable immune biomarkers in glioblastoma. Notably, radiologic assessment of lateral ventricle contact by standard-of-care MRI may guide clinical trial design for immunotherapies in neuro-oncology.

IMMU-41. NEOADJUVANT PD-1 BLOCKADE INDUCES T CELL AND CDC1 ACTIVATION BUT FAILS TO OVERCOME THE IMMUNOSUPPRESSIVE TUMOR ASSOCIATED MACROPHAGES IN RECURRENT GLIOBLASTOMA

Abstract In a small phase I clinical trial, neoadjuvant anti-PD-1 (neo-aPD1) improved survival in glioblastoma patients (GBM) and was associated with increased interferon-γ-related gene expression and an associated decrease in cell cycle-related gene expression in the tumor. Despite this, neo-aPD1 was not curative. To better understand how neo-aPD1 alters the GBM tumor microenvironment and to discover new therapeutic axes, we used CyTOF and single-cell RNAsequencing to analyze the tumor-infiltrating immune populations of 70 GBM patients, 20 of whom had received neo-aPD1. In patients treated with neo-aPD1 the proportion and number of tumor-infiltrating T cells increased and these T cells had increased expression of the cytokines CCL5 and XCL1, which promote dendritic cell migration. Among the infiltrating T cells was a population of progenitor exhausted CD8+ T cells expressing the genes TCF, SLAM6, CCR7, IL7R, and GZMB. Single-cell TCR analysis revealed that these CD8+ T cells arose from a population of early activated, cytotoxic T cells that had expanded in the peripheral blood and trafficked into the tumor. Within the tumor-infiltrating myeloid cells, we found that neo-aPD1 upregulated genes in the interferon-γ response pathways; namely genes related to T cell trafficking and immune suppression, such as CD274 (PD-L1), TREM2, GPNMB, and IL10, indicating a maladaptive response by some myeloid cells to the immune response triggered by neo-aPD1. Finally, we identified a new migratory and interferon-γ activated tumor-infiltrating conventional dendritic cell population that was increased with neo-aPD1 and that notably expressed XCR1, the receptor for XCL1. Additionally, our results provide future basis to study if modalities that enhance the T cell:DC axis or that inhibit the inhibitory myeloid populations may improve survival in patients with GBM when used in conjunction with neo-aPD1. In sum, we characterized the immune landscape in GBM and how it changes with neo-aPD1 at single cell resolution.

Combined BRAF, MEK, and CDK4/6 Inhibition Depletes Intratumoral Immune-Potentiating Myeloid Populations in Melanoma.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

The role of CD36 in macrophage lipid metabolism and function in tumor microenvironment

Abstract Macrophages are a highly plastic population of innate immunity with diverse functions in homeostasis. Despite their relative abundance in tumor microenvironment (TME), the exact function of tumor-associated macrophages (TAMs) and how they are regulated remain largely unknown. Macrophages also play key roles in lipid metabolism, disruption of which can result in pathologies such as atherosclerosis. Interestingly, TME has also been characterized as a lipid-rich context, we are interested in understanding how lipids in TME affect TAMs’ pro- or anti-tumor function. Here, we firstly characterized the lipid metabolic phenotype of TAMs in a mouse melanoma model. We found that up-regulation of lipid storage and uptake is associated with the most tolerogenic TAM subset marked by high level of F4/80. We also found high expression of scavenger receptor CD36 on F4/80hi TAMs which mediates uptake of oxidized LDL (oxLDL) from TME. Using germ line knockout (Cd36−/−) and myeloid-specific deficiency (Cd36 flox/flox x Csf1r-Cre) mouse models, we observed that uptake of oxLDL by TAMs was significantly blocked, along with rescued immunosuppressive phenotype characterized by decreased expression of PD-L1, CD206 and elevated secretion of TNFa, which together contribute to slower tumor growth. We further conducted single-cell RNA sequencing on Cd36−/− and wild-type TAMs, and found several pathways as potential mechanisms for CD36-mediated pro-tumor phenotype. In conclusion, we discovered that high expression of CD36 on TAMs mediates uptake of oxLDL and contribute to pro-tumor function. Our data suggests that metabolites in TME may play crucial roles in metabolic and functional reprogramming of tumor-infiltrating immune populations.

Phenotyping of tumor infiltrating immune cells using mass-cytometry (CyTOF).

The anti-tumor immune response plays a crucial role in cancer patient outcome as well as in response to the growing family of immunotherapeutic treatments. Improving patient prognostic and therapeutic management requires a better knowledge of the tumor microenvironment, for which a deep characterization of tumor-infiltrating immune populations is instrumental. Mass cytometry represents an excellent tool in tumor Immunology, as it allows the simultaneous analysis of >40 markers on single cells. In this chapter, we review challenging technical aspects of the mass cytometry phenotyping of tumor infiltrating immune cells, focusing on fresh human solid tumor samples. We first explain how to design mass cytometry experiments, then provide detailed protocols to isolate mononuclear immune cells from solid tissues and to stain them for an acquisition on a mass cytometer. We also discuss how to optimize the preparation of single immune cell samples, and how to ensure the reproducibility of data generated from distinct fresh human samples. Finally, we provide troubleshooting suggestions for difficult sample acquisitions on a mass cytometer.

Abstract 4969: TIGIT pathway phenotyping sheds light on promising strategies to restore anti-tumor immunity

Abstract TIGIT is a T cell co-inhibitory receptor recently described as a key checkpoint driving tumor cell immunosuppression. It is predominantly expressed on CD4+ Tregs, CD8+ T cells and NK cells from healthy individuals but further upregulated in cancer patients where it frequently co-expresses with exhaustion markers such as PD-1. TIGIT cognate receptors are members of the poliovirus receptors, among which CD155 has the highest affinity for TIGIT. They are expressed on antigen presenting cells but also on tumor cells which provides a strong rationale for blocking TIGIT as a therapeutic approach to reverse T or NK cell dysfunction linked with cancer progression. To substantiate the importance of TIGIT as an immunotherapy target, we used flow cytometry and immunohistochemistry (IHC) to extensively characterize TIGIT and CD155 expression in both healthy donors and cancer patients. We initially confirmed TIGIT expression on multiple immune subsets from healthy donors. Similar flow cytometry analysis performed on matched circulating and tumor-infiltrating immune populations from 15 cancer patients highlighted the global overexpression of TIGIT associated with cancer. Interestingly, ex vivo polyfunctional analysis of cytokine release strongly supported the immunosuppressed character of infiltrated TIGIT positive CD4+ and CD8+ T cells. Finally, combining receptor density with frequency of positive cells led to the interesting observation that tumor-infiltrating Tregs represents the population with the highest TIGIT expression, confirming the opportunity to preferentially target that suppressive population within the tumor microenvironment. These findings on Tregs were further confirmed by IHC. TIGIT was expressed in 10 out of the 11 lung adenocarcinoma tissues analysed. Both Tregs and CD8+ T cells express TIGIT, with Tregs showing a higher proportion of TIGIT+ cells. Finally, direct expression of TIGIT on tumor cells was also found on several haematological malignancies, opening the door for anti-TIGIT therapies to act directly on tumor cells besides revigoration of the immune system. The presence of TIGIT ligands within the tumor microenvironment was also assessed and confirmed using both flow cytometry and IHC. CD155 expression was analysed by IHC in both normal individuals (n=90) and cancer patient tissues from 9 different cancer indications (n=284). In cancer tissues, CD155 is mostly expressed by tumor cells, ranging from a median percentage of 50 % for pancreatic cancer to 2 % for cervix cancer. Cancer samples with the highest CD155 expression are pancreatic, prostate, kidney, gastric and colon cancers. CD155 expression is always lower in normal tissues compared to their cancer counterparts except for lung. Together, these data strongly support the relevance of targeting TIGIT in immuno-oncology and pave the way to select the ideal indications where patients could benefit from such a therapy. Citation Format: Noémie Wald, Marjorie Mercier, Julia Cuende, Florence Nyawouame, Shruthi Prasad, Margreet Brouwer, Erica Houthuys, Anne Marie-Cardine, Martine Bagot, Grégory Driessens, Véronique Bodo, Catherine Hoofd. TIGIT pathway phenotyping sheds light on promising strategies to restore anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4969.

Abstract 2346: Targeting the tumor immune response for pro-senescence therapy in prostate cancer

Abstract Cellular senescence is a stable growth arrest that constrains cell cycle progression in case proliferation becomes aberrant, thus limiting tumorigenesis. Oncogenic mutations and loss of tumor suppressor genes that cause uncontrolled cell-cycle progression are required events in tumor development. Nevertheless, many of these genetic alterations retain an intrinsic tumor-suppressive potential that gives rise to oncogene-induced senescence and tumor suppressor loss-induced senescence. Interestingly, many evidences support the idea that a strong interplay occurs between senescence, cancer cells, and tumor infiltrating immune populations, thus opening at the possibility to tune such interplay for cancer treatment. Indeed, tumor infiltrating T helper 1 cells can drive senescence in cancers in a cytokine-induced manner, by secreting IFNγ and TNFα in the tumor microenvironment. In addition, we recently described a model where Pten loss induced senescence is hindered in prostate tumor by non-cell-autonomous mechanisms. Our study support a model where tumor-infiltrating CD11b+Gr-1+ myeloid cells paracrinally triggers tumor senescence evasion in the prostate of Pten null mice, eventually promoting tumor progression. These findings we’ll be discussed. Interestingly, we will present new evidences from our lab indicating that M1 macrophages are polarized to the M2 phenotype in both Pten pc-/- and that treatments that interfere with the M1-M2 polarization and switch the skewing to the M1 polarization profile can induce senescence in Pten; Trp53pc-/- invasive prostate cancer tumours thereby arresting tumour progression. These findings open at the possibility that M1 directly control senescence in tumor cells. Finally, we have identified a gene expression signature that distinguishes myeloid infiltration in tumors and can be used as a predictive marker for tumor progression in prostate cancer patients. These evidences we’ll be also discussed. Citation Format: Diletta Di Mitri, JingJing Chen, Andrea Alimonti. Targeting the tumor immune response for pro-senescence therapy in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2346. doi:10.1158/1538-7445.AM2015-2346

Loss of MyD88 leads to more aggressive TRAMP prostate cancer and influences tumor infiltrating lymphocytes.

The influence of pattern recognition receptor (PRR) signaling in the prostate tumor microenvironment remains unclear. Although there may be a role for PRR agonists as adjuvants to therapy, prior evidence suggests tumor promoting as well as tumor inhibiting mechanisms. The purpose of this study is to examine the role of the key Toll-like receptor (TLR) signaling adaptor protein myeloid differentiation primary response gene 88 (MyD88) in prostate cancer development. MyD88(-/-) mice in a C57Bl6 background were crossed with transgenic adenocarcinomas of the mouse prostate (TRAMP) mice to create MyD88(-/-) TRAMP(Tg+/-) animals, which were compared to MyD88(+/+) TRAMP(Tg+/-) animals and their non-transgenic counterparts at 30 weeks. Prostates were examined histologically, by immunohistochemistry and immunofluorescence staining, and by qPCR, to characterize tumor-infiltrating immune populations as well as activation of the downstream NF-κB pathway and androgen receptor (AR) expression. Splenocytes were examined for development of distinct immune cell populations. Absence of MyD88 led to increased prostatic intraepithelial neoplasm (PIN) and areas of well-differentiated adenocarcinoma in TRAMP transgenic mice. Analysis of infiltrating immune populations revealed an increase in CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs), as evidenced by increased expression of prostatic arginase-1 and iNOS as well as the cytokine IL-10, and a deficiency in NK cells in prostates from MyD88(-/-) TRAMP(Tg+/-) compared to MyD88(+/+) TRAMP(Tg+/-) mice, whereas a decrease in splenocytic NK cell differentiation was observed in MyD88(-/-) mice. Prostate tumors revealed no significant differences in NF-κB or AR expression in MyD88(+/+) TRAMP(Tg+/-) compared to MyD88(-/-) TRAMP(Tg+/-) mice. During prostate cancer development in the TRAMP model, MyD88 may play a role in limiting prostate tumorigenesis by altering tumor-infiltrating immune populations. This suggests that in the context of specific cancers, distinct PRRs and signaling pathways of innate immune signaling may influence the tumor microenvironment and represent a novel therapeutic strategy.

Methods for Analysis of the Immune System in Mouse Cancer Models

Leukocytes of both the innate and adaptive immune systems can be a prominent feature of the tumor microenvironment. Mouse models of cancer that allow for tumor development in a host with a competent immune system can be investigated to gain a richer understanding of cancer immunobiology. Such studies not only provide biological insight but also can help forge new strategies for therapy and prognostication of patients. Here, we introduce several experimental protocols designed to analyze the immune phenotype of mouse cancer models. These include flow cytometric approaches to define tumor-infiltrating immune populations with precision and tissue-based assessments that preserve and reveal spatial dimensions of host-tumor cell interactions. An approach to study the immune-suppressive capability of myeloid cells in the tumor microenvironment is also provided.

Abstract B79: Tumor rejection driven by efficient Treg depletion is associated with a strong change in the tumor microenvironment.

Abstract Efficient regulatory T cell (Treg) depletion leads to tumor rejection in mice. The goal of this study was to understand the mechanisms involved in this rejection process. For the selective and efficient depletion of Tregs, we used the FoxP3.LuciDTR-4 mouse model where Tregs express, among others, the human diphtheria toxin receptor. Our previous studies show that the depletion of more than 90% of Tregs in FoxP3.LuciDTR-4 mice promotes the infiltration of immune cells and subsequent rejection of ovalbumin (OVA)-expressing B16 melanomas. To unravel the mechanisms involved, we characterized the tumor-infiltrating immune populations and performed a cytokine profile on tumors at the onset of rejection. The analysis revealed a change in the population composition of the tumor-infiltrating leucocytes. There was an increase in T cell numbers, especially CD8+ T cells, and dendritic cells in tumors in rejection. Moreover, the amount of B cells was drastically reduced in tumors being rejected while they were enriched in tumors in progression. Tumor-infiltrating myeloid cells also differed phenotypically between tumors in rejection and tumors in progression. Inflammatory cytokines related to the type 1 response such as IFN-γ, TNF and IL-12 were significantly more expressed in tumors in rejection as measured at mRNA and protein levels. On the other hand, the expression of some cytokines related to the type 2 response such as IL-5 and IL-10 and of the inhibitory cytokine TGF-β did not change between tumors in rejection and in progression. We also determined that the concentration of molecules related to the M1 macrophage phenotype such as inducible nitric oxide synthase (iNOS), interferon regulatory factor 5 (IRF5), CXCL9 and CXCL10 was significantly higher in tumors in rejection; however, the concentration of most of the molecules related to the M2 macrophage phenotype such as CD163, macrophage mannose receptor 1 (MRC1), Fizz-1 and PPAR-γ was not different between tumors in rejection and tumors in progression. Interestingly, angiogenesis drivers like VEGF and angiotensin 1 (ANG1) were found to be significantly less expressed in tumors in rejection, a fact that correlates with the normalization of the tumor vasculature previously described to be promoted by Treg depletion. We conclude that Treg depletion provokes a strong change in the tumor microenvironment, not only in terms of the elevated number of immune infiltrating cells but also in terms of the enrichment of certain subpopulations. Furthermore, this change is associated with a striking alteration of the cytokine and chemokine profile that becomes proinflammatory, supports mainly a Type 1-related response and inhibits angiogenesis pathways. Citation Format: Oscar C. Salgado, Rafael Carretero, Xingrui Li, Ibrahim M. Sektioglu, Natalio Garbi, Günter J. Hämmerling. Tumor rejection driven by efficient Treg depletion is associated with a strong change in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B79.