Choreographies of nearness: self and other in personal cancer immune therapy research

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Background Intravital multiphoton microscopy (IMM) provides single cell imaging within intact living systems. IMM of the autofluorescent metabolic co-enzymes NAD(P)H and FAD, optical metabolic imaging (OMI), provides in vivo label-free...

BackgroundThe clinical outcomes are not always favorable in certain thyroid cancer patients. The effect of Forkhead-box family on immune cells infiltration and tumor microenvironment in thyroid cancer was explored. The role of FOXP2 in tumor invasion and recurrence was investigated consequently.MethodsTIMER and GEPIA were firstly employed to compare FOXPs expression in normal and cancer tissues from multiple human cancers. The results from database were confirmed by quantitative Real Time-PCR and Western blot in matched thyroid cancer and adjacent normal tissues, in addition to a panel of thyroid cancer cell lines and normal thyroid cell. GEPIA platform was employed to discover the possibility of FOXPs as prognostic indicator. TISIBD and UACLCAN were then employed to estimate the influence of FOXPs on lymph node metastasis and tumor staging. GEPIA analysis was initially employed to analyze correlation of FOXPs and tumor immune infiltrating cells, and TIMER dataset was then included for standardization according to tumor purity.ResultDifferent member of FOXPs showed divergence in expression in various cancer tissues. Lower FOXP1, FOXP2 and higher FOXP3, FOXP4 levels could be identified in thyroid cancer tissues when compared with matched normal tissue. There was an inverse correlation between FOXP2, FOXP4 and immune invasion, whereas FOXP1 and FOXP3 were positively correlated. FOXPs showed remarkable correlations with multiply immune cells. More importantly, only FOXP2 showed the significant effect on recurrence and tumor staging.ConclusionAs immune regulatory factor, the reduction of FOXP2 may affect tumor microenvironments and immune cells infiltration, enhance tumor immune escape, and promote recurrence of thyroid cancer. FOXP2 could be a new potential diagnostic and prognostic marker.

Impacts of transmembrane serine protease 4 expression on susceptibility to severe acute respiratory syndrome coronavirus 2.

AIMTo investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing.METHODSHuman iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer (OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer.RESULTSIn eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells.CONCLUSIONThe genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

Current chemotherapy and radiation therapies for hepatocellular carcinoma (HCC), which causes 500,000 deaths per year worldwide, are not very effective. The recently approved multikinase inhibitor Sorafenib modestly improves survival by a few months in some patients with advanced cancer (1). Surgical resection and liver transplantation can be performed in less than 20% of patients. Thus, development of novel diagnostic and prognostic approaches and therapeutic modalities for HCC is urgently needed. HCC is unique compared with other human cancers because the majority of HCC occurs in patients with chronic liver diseases, especially viral hepatitis (ie, hepatitis B and hepatitis C) and liver cirrhosis due to various etiologies (2). The latent period from the original disease to the development of HCC usually ranges from 10 to 30 years; this long window of time provides an opportunity for investigating cancer development and designing strategies for intervention. To achieve this end, a better understanding of the mechanisms by which HCC develops and progresses is critically important. The common denominator underlying HCC carcinogenesis is inflammation, a form of host immune defense. It is most obvious in the setting of viral hepatitis, especially hepatitis C viral infection. Hepatitis C virus is the major etiological factor for HCC in most developed countries. Hepatitis C virus has an ability to evade its host’s innate and adaptive immune system and establish persistent infection, resulting in lasting inflammatory response in the liver (3). Although the detailed molecular cascades are not entirely known, there is no doubt that inflammation can cause genetic and epigenetic dysregulation in hepatocytes and, eventually, cell transformation. Once cancer is established in the liver, the host’s innate and adaptive immune system still persistently attempts to eradicate or slow the cancer cell growth. Recent studies have demonstrated that the vigor and breadth of the immune system play a critical role in the outcome of HCC (4,5). However, the mechanism of how various components of the immune system interact with HCC remains a mystery. The innate immune response against cancer is important because it not only directly attacks cancer cells but also primes specific and long-lasting adaptive immune response. In HCC patients, antigen-specific T cells are detectable, which indicates that both innate and adaptive immune responses are operative in HCC. Despite this immune response, cancer still prevails. One explanation for the insufficient immune surveillance is that immunosuppressive function mediated by regulatory T cells and myeloid-derived suppressor cells is upregulated in HCC patients (6–8). The increased infiltration of these immunosuppressive cells creates an immunosuppressive milieu in tumor tissue, which is associated with a bleak clinical prognosis. Studies showed that modulation of regulatory T cells improved overall antitumor immunity (9,10). Whether this strategy can be successfully used in HCC immunotherapy remains to be investigated. In addition, there are many other immune cells that infiltrate HCC cancer tissue, such as natural killer (NK) cells, macrophages, and different subsets of T and B cells. These cells can produce numerous cytokines that are almost certain to affect the tumor microenvironment. Moreover, HCC cells are capable of making molecules that counter the host immune response (10). Understanding the tumor microenvironment will undoubtedly help us figure out how to shift a tumor-friendly environment to a host-friendly one. When discussing inflammatory reactions, a well-known proinflammatory family of proteins cannot be ignored. Initially considered key receptors to sense pathogens and activate the innate immune response against bacterial, viral, and fungal infection, Toll-like receptors (TLRs) have been recently recognized to play a role in cancer (11). Expression of different members of TLRs has been reported in a number of human cancers (12). However the functional role of TLRs is much more complicated. Both protumor growth and antitumor activities were reported (13). Clearly more experiments are needed to unravel the biological role of TLRs in cancer. More important, we need to find out how to use the knowledge for cancer therapy because multiple TLR agonists have been developed (14). In this issue of the Journal, Chew et al. present their intriguing study on the role and the underlying mechanism of TLR3 in HCC (15). This study brings attention to the effect of innate immunity on HCC progression and its clinical outcome. The investigators examined the expression levels of TLR3 in 172 human liver cancer tissues from predominantly female (82%) patients and found that increased TLR3 expression is positively correlated with a longer survival for HCC patients. Using immunohistochemical staining, they identified TLR3 overexpression in both HCC cancer cells and tumor-infiltrating NK cells and, independently, that higher expression of TLR3 on either cell population is associated with longer survival. The investigators then performed a series of experiments to demonstrate that activation of TLR3 by polyinosinic:polycytidylic acid (poly I:C) caused apoptosis of the TLR3-positive HCC cell lines, whereas similarly activated TLR3 promoted NK cell proliferation and increased NK cell antitumor activity. The investigators further examined the impact of TLR3 activation on cancer cells and immune cells in mouse models. Similar to the in vitro observation, they found that both T cells and NK cells proliferated upon poly (I:C) treatment, whereas in vivo the HCC cells exhibited fourfold lower proliferation and an 11-fold increase in tumor cell apoptosis. The findings suggest that effects of TLR3 are quite different in immune cells and in cancer cells. The tantalizing questions are how TLR3 knows how to act differently in normal and cancer cells and what the downstream signaling molecules are. Chew et al. also examined the effect of TLR3 activation in murine HCC models (15). When mice with endogenous HCC were treated with poly (I:C), there was increased expression of Ccl5 and CXCL9 in both the HCC cancer cells and tumor-infiltrating leukocytes. NK cell numbers also increased; these cells appeared to be activated in the tumors. The authors further demonstrated in a transplant HCC mouse model that the expression of Ccl5 and CXCL9 increased tumor-infiltrating lymphocytes and suppressed the tumor growth. The data seem to suggest that TLR3 may function through induction of these two cytokines. This interpretation may be an oversimplification of a much more sophisticated process. It is well known that TLR3 can activate interferon regulatory factor 3 (IRF3) and induce abundant production of type 1 interferons. One has to wonder what happens to interferons in the tumor and what role interferons play. It is a relevant question because interferons have an antitumor effect on many types of human cancers, including HCC, as well as an immune regulatory effect. One of the most interesting findings from this study is the demonstration of the multiple functions of TLR3: slowing cancer cell proliferation, increasing apoptosis, and attracting NK cells and T cells and enhancing their proliferation and antitumor effect. These functions are all beneficial for fighting cancer, at least in HCC. It is not surprising that TLR3 can cause HCC apoptosis because it has been shown before that TLR3 and IRF3 activation induces the production of tumor necrosis factor–related apoptosis-inducing ligand (16). The puzzle is why this TLR3-mediated pathway does not act earlier to stop the cancer in the first place. Because hepatitis C virus has an ability to interfere with TLR3 signaling (17), it is possible that cancer cells can act similarly. Although the molecular mechanisms remain to be defined, activation of TLR3 appears to be beneficial for HCC patients and raises the hope that TLR3 can be used as a target for immunotherapy. The use of TLR3 agonists in this study provides proof of concept. However, the immune system can be a double-edged sword, and we must proceed carefully with experimental therapy. The challenge is how to kill cancer cells without causing damage to noncancerous hepatocytes. The nontumor liver tissue has its own unique microenvironment created by interaction between viruses and immune responses, and we should focus on how to selectively turn on TLR3 in cancer tissues but not in the whole liver or the whole body. The timing, intensity, and location of certain immune functions are paramount to maintain homeostasis while fighting off disease. Understanding how TLRs or other innate immune components operate in the cancer microenvironment will certainly enhance our ability to fine-tune the immune system to fight cancer.

BackgroundInteractions between tumor and neighboring immune cells are dynamic and complex within the tumor microenvironment (TME), requiring the use of spatial biology techniques to characterize them, and thereby identify potential...

The article discusses modern ideas about the immune therapy of cancer — methods of treatment of oncological diseases based on immunological reactions of the organism to the appearance of malignant cells in it. This area is actively studied in clinical practice in the last decade, and some therapy has already been approved for use by regulators after promising results of clinical trials 3 phase.Immune therapy is based on antitumor immune cycle — the cascade of processes responsible for the immune system’s response to tumor cells. Involved regulatory mechanisms are targets for various therapies, the overall goal is to restore proper functioning of the cycle and to achieve the elimination of cancer cells.Currently, the most studied two types of immune therapy — checkpoint inhibitors and adaptive cell therapy. Checkpoint inhibitors increase the activity of body immune cells, reducing the inhibitory influence of the tumor microenvironment and the tumor cells themselves, which allowed them to get out from under the pressure of the immune system during the development of the disease. Adaptive cell therapy, in turn, allows to compensate the lack of active immune against tumor cells.Mechanisms of action determine the effectiveness of various therapies for different diseases, and for patients inside of one diagnosis. To determine the effectiveness of other treatment prior to a particular patient it is necessary to use the latest achievements in precision medicine, based on the search for new biomarkers and analyzing each patient separately. This approach will significantly reduce costs and save precious time for the patient.

In recent years, advances in spatial transcriptomics have revolutionized our understanding of the tumor microenvironment, providing crucial insights into the complex interplay of different cell types within cancer tissues. In this study, we employed the new, high definition Visium spatial transcriptomics assay (Visium HD) to investigate the intricate molecular landscape of prostate cancer at single-cell scale resolution and across the whole transcriptome. Our research focused on deciphering the spatial heterogeneity of gene expression patterns within the tumor microenvironment, shedding light on the interactions between cancer cells, stromal cells, and vasculature. The Visium HD assay enables an unbiased exploration of the transcriptome on FFPE tissue sections mounted on standard glass slides. Combining gene expression data with H&E images from the same section, it allows precise characterization of local molecular features and disease states at single-cell scale. We analyzed a set of human prostate adenocarcinoma samples with the Visium HD assay, and identified spatially regulated gene signatures associated with specific cell types and functional pathways. Our findings have recapitulated previously identified molecular markers, including VEGFA and MYC which have been implicated in tumor progression and angiogenesis. The spatial gene expression patterns had good correlation with histological annotations of cancerous and normal tissue within the H&E image. However, Visium HD allowed us to further observe molecular processes and pathways specific to this adenocarcinoma sample. This integrative approach offers valuable implications for personalized cancer therapy and the development of targeted interventions tailored to the spatial context of individual tumors. Our study underscores the significance of understanding the spatial organization of the whole transcriptome in cancer tissues, and highlights the potential of the Visium HD platform as a powerful tool for unraveling the complexities of the tumor microenvironment. These insights pave the way for the development of innovative therapeutic strategies and precision medicine approaches, ultimately contributing to improved outcomes for cancer patients. Citation Format: Yifeng Yin, Jerald Sapida, David Sukovich, David Patterson, Augusto Tentori. Unraveling spatial complexity of the tumor microenvironment: A whole transcriptomic perspective with Visium HD [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 3645.

BackgroundBladder cancer (BC) is the 10th most common cancer world-wide with an estimated 570,000+ people being diagnosed in 2020.1 It has been shown that communities of bacteria (bacterial microbiome) exist...

Colon cancer represents a significant malignant neoplasm within the digestive system, characterized by a high incidence rate and substantial disease burden. The F-box protein 22 (FBXO22) plays a role in forming a specific type of ubiquitin ligase subunit, which is expressed abnormally in various malignant neoplasms and shows a notable relationship with prognosis in patients with cancer. Nevertheless, the function of FBXO22 in the context of colon cancer remains inadequately elucidated. To explore the role of FBXO22 in colon cancer by examining FBXO22 expression patterns and analyzing how the protein affects the prognosis in patients who have undergone surgery. Samples of cancerous and nearby normal tissues from patients with colon cancer were gathered, along with pertinent clinical data. Expression levels of the FBXO22 gene in both cancerous and paracancerous tissues were assessed through immunohistochemistry. The median H score served as a criterion for categorizing FBXO22 gene expression into high and low levels in cancerous tissues, and the relationship between these expression levels and various pathologic characteristics of patients, such as age, sex, and clinical stage, was analyzed. Colon cancer cell lines HCT116 and DLD-1 were used and divided into three groups: A blank control group, a negative control group, and a si-FBXO22 group. FBXO22 gene mRNA and protein expression were measured 24 hours post-transfection using real-time fluorescence quantitative polymerase chain reaction and western blotting. The proliferation capabilities of the cells in each group were assessed using the Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine assay, while cellular migration and invasion abilities were evaluated using scratch healing and Transwell assays. Various online platforms, including the Timer Immune Estimation Resource, were used to analyze pan-cancer expression, promoter methylation levels, and mutation frequencies of the FBXO22 gene in colon cancer patients. Additionally, the correlation between FBXO22 gene expression, patient prognosis, immune cell infiltration, and the expression of immune molecules in the colon cancer microenvironment was investigated. The relationship between FBXO22 gene expression and chemotherapy resistance, along with the potential mechanisms of action of the FBXO22 gene, were analyzed using The Cancer Genome Atlas dataset and the Genomics of Drug Sensitivity in Cancer drug training set via R software. Compared with normal colonic tissues, the FBXO22 gene was highly expressed in colon cancer tissues. Post-operative patients with colon cancer elevated FBXO22 reduced survival and exhibited resistance to various chemotherapeutic agents. FBXO22 expression suppresses the infiltration of anti-tumor immune cells. In vitro, FBXO22 knockdown inhibited the proliferation and migration of colon cancer cells. The FBXO22 gene is a biomarker of poor prognosis in patients with colon cancer and has potential as a target for immunotherapy and overcoming chemotherapy resistance.

Current cancer biology acknowledges the key role of the immune system in tumor biology, and promise for the modulation of immune system in cancer treatment. The composition of the inflammatory cell populations in tissues is reflective of the overall state of the Tumor Micro-Environment (TME), and the identification of distinct inflammatory cell types may hold prognostic or predictive value. Immunohistochemistry allows for reliable identification of the cell constituents to facilitate analysis of the TME while remaining in the tissue context. Establishing a quantitative paradigm for inflammatory cell types and subtype profiling requires unbiased and automated whole-tissue based quantitation methods, which are capable of spatial integration of multiple inflammatory cell markers across the whole tissue. While single slide fluorescent multiplex approaches can address this need, the use of difficult-to-implement wet assay strategies involving multiplexing 6-8 fluorescent markers on the same tissue section are difficult to implement in a global clinical diagnostic lab setting. To answer this need, we combined novel advents in Tissue Image Analysis (TIA) to integrate spatial expression of serial-section stained whole tissue clinical lung cancer specimens. In this proof-of-principle study,we were able to superimpose specific locations of individual cell types onto 6 serial sections and evaluate different inflammatory cell types. We used serial sections of clinical lung specimens stained for six immune phenotypic markers (CD68, CD4, CD8, CD33, FoxP3, and CD11b) to illustrate a repertoire of inflammatory cell types. Our proprietary CellMap algorithm was utilized to identify, enumerate, and determine the precise location of individual inflammatory cells in tissues on cell-by-cell basis in the tumor microenvironment (TME). Our proprietary FACTS (Feature Analysis on Consecutive Tissue Sections) approach was used to integrate the spatial expression of individual markers onto a reference H&E slide, and/or adjacent slides. Using the aligned FACTS data and our proprietary MultivariateMap approach, we integrated the patterns of each marker based on immune cell type function and their location relative to each other and the tumor epithelial cells. In this study, we demonstrated how spatial integration of immune cell markers in the context of whole tissues can be applied to the diagnostic setting. By creating a comprehensive landscape of the immune system state in the tissue biopsies, we were able to identify crucial patterns which represent function and role in immune system biology. These approaches provide a robust platform for immuno-oncology applications by providing information on the state of the immune system in cancer using approaches implementable in the clinic. The use of these approaches will benefit further understanding of cancer pathology, and can directly lead to the development of diagnostic tests with clinical utility. Citation Format: Joseph S. Krueger, Nathan Martin, Famke Aeffner, Anthony Milici, John Alvarez, Micheal Sharp. Quantitative analysis of multiple subtypes of immune system cells in cancer tissues. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C109.

Current cancer biology acknowledges the key role of the immune system in tumor biology, and promise for the modulation of immune system in cancer treatment. The composition of the inflammatory cell populations in tissues is reflective of the overall state of the Tumor Micro-Environment (TME), and the identification of distinct inflammatory cell types may hold prognostic or predictive value. Immunohistochemistry allows for reliable identification of the cell constituents to facilitate analysis of the TME while remaining in the tissue context. Establishing a quantitative paradigm for inflammatory cell types and subtype profiling requires unbiased and automated whole-tissue based quantitation methods, which are capable of spatial integration of multiple inflammatory cell markers across the whole tissue. While single slide fluorescent multiplex approaches can address this need, the use of difficult-to-implement wet assay strategies involving multiplexing 6-8 fluorescent markers on the same tissue section are difficult to implement in a global clinical diagnostic lab setting. To answer this need, we combined novel advents in Tissue Image Analysis (TIA) to integrate spatial expression of serial-section stained whole tissue clinical lung cancer specimens. In this proof-of-principle study,we were able to superimpose specific locations of individual cell types onto 6 serial sections and evaluate different inflammatory cell types. We used serial sections of clinical lung specimens stained for six immune phenotypic markers (CD68, CD4, CD8, CD33, FoxP3, and CD11b) to illustrate a repertoire of inflammatory cell types. Our proprietary CellMap algorithm was utilized to identify, enumerate, and determine the precise location of individual inflammatory cells in tissues on cell-by-cell basis in the tumor microenvironment (TME). Our proprietary FACTS (Feature Analysis on Consecutive Tissue Sections) approach was used to integrate the spatial expression of individual markers onto a reference H&E slide, and/or adjacent slides. Using the aligned FACTS data and our proprietary MultivariateMap approach, we integrated the patterns of each marker based on immune cell type function and their location relative to each other and the tumor epithelial cells. In this study, we demonstrated how spatial integration of immune cell markers in the context of whole tissues can be applied to the diagnostic setting. By creating a comprehensive landscape of the immune system state in the tissue biopsies, we were able to identify crucial patterns which represent function and role in immune system biology. These approaches provide a robust platform for immuno-oncology applications by providing information on the state of the immune system in cancer using approaches implementable in the clinic. The use of these approaches will benefit further understanding of cancer pathology, and can directly lead to the development of diagnostic tests with clinical utility. Citation Format: Mirza Peltjo, Carsten Schnatwinkel, Nathan Martin, Holger Lange, Joseph S. Krueger. Quantitative analysis of multiple subtypes of immune system cells in cancer tissues. [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 2360. doi:10.1158/1538-7445.AM2015-2360

Immune checkpoint inhibitor therapy (ICT) can provide durable remissions for cancer patients with previously incurable malignancies. However, ICT is ineffective for many cancer types, and even those who initially respond may develop ICT resistance and succumb to their cancer. Tumor cell extrinsic factors, termed host factors, have recently emerged as key mediators and biomarkers of ICT response. One intriguing host factor that may influence ICT response is the circadian clock, an evolutionarily conserved transcriptional-translational feedback loop that allows mammalian organisms to adapt to environmental exposures (e.g. light). However, whether “the clock” modulates the ICT anti-tumor immune response is unknown. Utilizing preclinical mouse models of cancer immunotherapy, we found that ICT efficacy is time-of-day dependent. ICT was most efficacious if ICT was administered at two hours after first light exposure (Zeitgeber time 2, ZT2) and least efficacious at ZT18. Utilizing flow cytometry, bulk cytokine analysis, and single cell RNA sequencing, we characterized the anti-tumor immune response after administering ICT at ZT2 and ZT18. There was an increase in effector granzyme B- and interferon-γ producing CD8+ T-cells in the tumor at ZT2 versus ZT18. This was accompanied by an increase in CD11c+ dendritic cells (DCs) and intratumoral chemokines (e.g. CXCL10) that promote DC/T-cell migration into the tumor microenvironment, and leads to enhanced DC-T-cell priming and anti-tumor immunity. Interestingly, these time-of-day dependent differences in tumor immune cell infiltration were present prior to the administration of ICT. To interrogate the role of the clock in mediating these time-of-day dependent differences, we characterized the response to ICT in mice that lack the core clock gene, BMAL1. Loss of BMAL1 in DCs (BMAL1fl/fl crossed with CD11c-cre) attenuated ICT efficacy, abrogated time-of-day dependent differences in ICT efficacy and dampened anti-tumor immunity. These findings highlight a role for a novel host factor, the circadian clock, in mediating the efficacy of cancer immune checkpoint therapy. Citation Format: Jake N. Lichterman, Tarun Srinivasan, Wenling Li, Parastoo Sabaeifard, Laura A. Coughlin, Nicole Poulides, Lora V. Hooper, Andrew Y. Koh. The circadian clock modulates cancer immune checkpoint therapy through immune cell trafficking [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 2658.