Later Stages Of Tumor Progression Research Articles

Abstract 6208: Characterization of molecular evolution in myelodysplastic neoplasms

Abstract Myelodysplastic neoplasms (MDS) are hematopoietic stem cell disorders. The IPSS-Mol model, a scoring system that combines molecular and clinical features to stratify MDS patients, can identify individuals at high-risk of developing Acute Myeloid Leukemia (AML), enabling the targeted application of disease-modifying therapies. However, the molecular evolutionary patterns in MDS and their impact on Overall Survival (OS) and Leukemia-Free Survival (LFS) remain unexplored. Shifting from conventional static mutational analyses used by IPSS-Mol to a dynamic evolutionary approach, we employed the ASCETIC framework to analyze data from the IPSS-Mol Database (3,323 patients, 152 genes) and understand the dynamics of MDS progression. According to ASCETIC, genes related to DNA methylation displayed low ranks, indicating their tendency to manifest early in the progression of the disease. In contrast, genes associated with cellular signaling typically appeared in the later stages of tumor progression, while genes involved in RNA splicing exhibited intermediate ranks.Regarding molecular evolution in MDS, ASCETIC identified five clusters, each characterized by distinct evolutionary signatures and significantly different OS and LFS rates (p < 0.001). The median OS for the C1, C2, C3, C4, and C5 clusters were 79.3, 64.1, 28.9, 16.2, and 14.8 months, respectively. Subsequently, we compared ASCETIC's clusters with the different risk groups defined by the IPSS-Mol score. Among the 1158 patients initially classified as V-Low/M-Low risk by IPSS-Mol, ASCETIC identified 55 patients placed in C4 and C5. These patients exhibited lower OS (p = 0.005) and LFS (p = 0.02). Conversely, among the 905 IPSS-Mol M-High/V-High risk patients, ASCETIC identified 468 patients (51.7%) classified as low risk (C1-C2-C3), who were associated with longer median OS and LFS (p < 0.001). The differences between conventional mutational analyses and our evolutionary approach primarily, but not exclusively, arise from the evaluation of TP53 mutations and ASXL1. In the IPSS-Mol model, a higher risk is conferred if multi-hit TP53 alterations occur or in the presence of ASXL1 mutations.Conversely, ASCETIC assesses the impact of mutations differently. It considers not only the Variant Allele Frequencies of individual- and/or co-mutations but also the evolutionary trajectories that lead to TP53 and/or ASXL1. This approach results in different risk weights assigned to each evolutionary trajectory.Lastly, we identified patients with NPM1 mutations, classified as MDS patients in the original dataset but now categorized as AML according to 2022-WHO. Within this high-risk context, ASCETIC differentiated between patients with shorter and longer Overall Survival (OS) (p < 0.001).Our study reveals the influence of evolution on MDS prognosis, offering insights into the integration of the ASCETIC framework with the IPSS-Mol. Validation on external cohort is ongoing. Citation Format: Ivan Civettini, Valentina Crippa, Federica Malighetti, Matteo Villa, Andrea Aroldi, Fabrizio Cavalca, Alex Graudenzi, Lorenza Maria Borin, Luca Mologni, Rocco Piazza, Carlo Gambacorti-Passerini, Daniele Ramazzotti. Characterization of molecular evolution in myelodysplastic neoplasms [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 6208.

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer [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 287.

Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Cooperative pro-tumorigenic adaptation to oncogenic RAS through epithelial-to-mesenchymal plasticity.

In breast cancers, aberrant activation of the RAS/MAPK pathway is strongly associated with mesenchymal features and stemness traits, suggesting an interplay between this mitogenic signaling pathway and epithelial-to-mesenchymal plasticity (EMP). By using inducible models of human mammary epithelial cells, we demonstrate herein that the oncogenic activation of RAS promotes ZEB1-dependent EMP, which is necessary for malignant transformation. Notably, EMP is triggered by the secretion of pro-inflammatory cytokines from neighboring RAS-activated senescent cells, with a prominent role for IL-6 and IL-1α. Our data contrast with the common view of cellular senescence as a tumor-suppressive mechanism and EMP as a process promoting late stages of tumor progression in response to signals from the tumor microenvironment. We highlighted here a pro-tumorigenic cooperation of RAS-activated mammary epithelial cells, which leverages on oncogene-induced senescence and EMP to trigger cellular reprogramming and malignant transformation.

Phosphorylated nuclear DICER1 promotes open chromatin state and lineage plasticity of AT2 tumor cells in lung adenocarcinomas.

KRAS/ERK pathway phosphorylates DICER1, causing its nuclear translocation, and phosphomimetic Dicer1 contributes to tumorigenesis in mice. Mechanisms through which phospho-DICER1 regulates tumor progression remain undefined. While DICER1 canonically regulates microRNAs (miRNA) and epithelial-to-mesenchymal transition (EMT), we found that phosphorylated nuclear DICER1 (phospho-nuclear DICER1) promotes late-stage tumor progression in mice with oncogenic Kras, independent of miRNAs and EMT. Instead, we observe that the murine AT2 tumor cells exhibit altered chromatin compaction, and cells from disorganized advanced tumors, but not localized tumors, express gastric genes. Collectively, this results in subpopulations of tumor cells transitioning from a restricted alveolar to a broader endodermal lineage state. In human LUADs, we observed expression of phospho-nuclear DICER1 in advanced tumors together with the expression of gastric genes. We define a multimeric chromatin-DICER1 complex composed of the Mediator complex subunit 12, CBX1, MACROH2A.1, and transcriptional regulators supporting the model that phospho-nuclear DICER1 leads to lineage reprogramming of AT2 tumor cells to mediate lung cancer progression.

Abstract A018: Phosphorylated nuclear DICER1 promotes open chromatin state and lineage plasticity of lung adenocarcinomas

Abstract DICER1 controls micro(mi)RNA-mediated epithelial-to-mesenchymal transition (EMT) to regulate tumorigenesis of lung adenocarcinomas (LUADs). We discovered that DICER1 is phosphorylated by ERK and nuclear translocated and phospho-DICER1 contributes to tumorigenesis. Mechanisms through which phospho-DICER1 regulates tumor progression remain undefined. We show that phospho-nuclear DICER1 associates with invasive human LUADs with oncogenic KRAS mutations and promotes late-stage tumor progression in mice with oncogenic Kras mutations. Surprisingly, phosphomimetic DICER1 regulates LUAD progression independent of miRNAs and EMT. Integrating single-cell RNA sequencing, fluorescent in situ RNA hybridization, immunofluorescence, and ATAC-sequencing, in mice,we discovered that phosphomimetic DICER1 generates an open chromatin state in the lung tumor alveolar cells leading to expression of gastrointestinal genes and altered AT2 cell identity. Strikingly, we also observe the gastric gene signature in human LUADs with phospho-DICER1 and KRAS mutations. We propose that phosphorylated nuclear DICER1 regulates chromatin remodeling leading to tumor cell reprogramming which drives lung cancer progression. Citation Format: Raisa A. Reyes-Castro, Shin-Yu Chen, Jacob Seemann, Swathi Arur. Phosphorylated nuclear DICER1 promotes open chromatin state and lineage plasticity of lung adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A018.

How is the AKT/mTOR pathway involved in cell migration and invasion?

As a pathway that plays a role in nutrient absorption, anabolic response, cell growth and survival, the important role of AKT/mTOR in tumorigenesis has also come to light. For cancer patients, most deaths are caused by the growth of metastatic tumors outside the primary focus. Therefore, migration and invasion in the late stage of tumor progression are the main unresolved issues in the study of tumor pathogenesis, and AKT/mTOR has been found to participate in the migration and invasion of cancer cells, which means that the study of this pathway may contribute to a solution for the problem. Because of its extensive and complex functions in the organism, this pathway can be regulated by a variety of different signals in the body, and then realize its function through different downstream signal molecules. This article reviews the proteins that can indirectly affect this pathway by regulating the common upstream signaling molecules of this pathway, and the proteins that can directly affect the level of phosphorylation of AKT/mTOR in cancer cells. We also review the proteins that can co-regulate this pathway and its downstream pathways. Through this study, we hope to gain a deeper understanding of the regulatory mechanism of the AKT/mTOR pathway in cancer cells, in hopes of finding effective and harmless cancer treatment targets in the future.

Implications of reactive oxygen species in lung cancer and exploiting it for therapeutic interventions.

Lung cancer is the second (11.4%) most commonly diagnosed cancer and the first (18%) to cause cancer-related deaths worldwide. The incidence of lung cancer varies significantly among men, women, and high and low-middle-income countries. Air pollution, inhalable agents, and tobacco smoking are a few of the critical factors that determine lung cancer incidence and mortality worldwide. Reactive oxygen species are known factors of lung carcinogenesis resulting from the xenobiotics and their mechanistic paths are under critical investigation. Reactive oxygen species exhibit dual roles in cells, as a tumorigenic and anti-proliferative factor, depending on spatiotemporal context. During the precancerous state, ROS promotes cancer origination through oxidative stress and base-pair substitution mutations in pro-oncogenes and tumor suppressor genes. At later stages of tumor progression, they help the cancer cells in invasion, and metastases by activating the NF-kB and MAPK pathways. However, at advanced stages, when ROS exceeds the threshold, it promotes cell cycle arrest and induces apoptosis in cancer cells. ROS activates extrinsic apoptosis through death receptors and intrinsic apoptosis through mitochondrial pathways. Moreover, ROS upregulates the expression of beclin-1 which is a critical component to initiate autophagy, another form of programmed cell death. ROS is additionally involved in an intermediatory step in necroptosis, which catalyzes and accelerates this form of cell death. Various therapeutic interventions have been attempted to exploit this cytotoxic potential of ROS to treat different cancers. Growing body of evidence suggests that ROS is also associated with chemoresistance and cancer cell immunity. Considering the multiple roles of ROS, this review highlights the exploitation of ROS for various therapeutic interventions. However, there are still gaps in the literature on the dual roles of ROS and the involvement of ROS in cancer cell immunity and therapy resistance.

Identification of Galectin-7 as a crucial metastatic enhancer of squamous cell carcinoma associated with immunosuppression.

Metastasis predicts poor prognosis in cancer patients. It has been recognized that specific tumor microenvironment defines cancer cell metastasis, whereas the underlying mechanisms remain elusive. Here we show that Galectin-7 is a crucial mediator of metastasis associated with immunosuppression. In a syngeneic mouse squamous cell carcinoma (SCC) model of NR-S1M cells, we isolated metastasized NR-S1M cells from lymph nodes in tumor-bearing mice and established metastatic NR-S1M cells in in vitro culture. RNA-seq analysis revealed that interferon gene signature was markedly downregulated in metastatic NR-S1M cells compared with parental cells, and in vivo NR-S1M tumors heterogeneously developed focal immunosuppressive areas featured by deficiency of anti-tumor immune cells. Spatial transcriptome analysis (Visium) for the NR-S1M tumors revealed that various pro-metastatic genes were significantly upregulated in immunosuppressive areas when compared to immunocompetent areas. Notably, Galectin-7 was identified as a novel metastasis-driving factor. Galectin-7 expression was induced during tumorigenesis particularly in the microenvironment of immunosuppression, and extracellularly released at later stage of tumor progression. Deletion of Galectin-7 in NR-S1M cells significantly suppressed lymph node and lung metastasis without affecting primary tumor growth. Therefore, Galectin-7 is a crucial mediator of tumor metastasis of SCC, which is educated in the immune-suppressed tumor areas, and may be a potential target of cancer immunotherapy.

Temporal radiographic and histological study of necrosis development in a mouse glioblastoma model.

Tumor necrosis is a poor prognostic marker in glioblastoma (GBM) and a variety of other solid cancers. Accumulating evidence supports that necrosis could facilitate tumor progression and resistance to therapeutics. GBM necrosis is typically first detected by magnetic resonance imaging (MRI), after prominent necrosis has already formed. Therefore, radiological appearances of early necrosis formation and the temporal-spatial development of necrosis alongside tumor progression remain poorly understood. This knowledge gap leads to a lack of reliable radiographic diagnostic/prognostic markers in early GBM progression to detect necrosis. Recently, we reported an orthotopic xenograft GBM murine model driven by hyperactivation of the Hippo pathway transcriptional coactivator with PDZ-binding motif (TAZ) which recapitulates the extent of GBM necrosis seen among patients. In this study, we utilized this model to perform a temporal radiographic and histological study of necrosis development. We observed tumor tissue actively undergoing necrosis first appears more brightly enhancing in the early stages of progression in comparison to the rest of the tumor tissue. Later stages of tumor progression lead to loss of enhancement and unenhancing signals in the necrotic central portion of tumors on T1-weighted post-contrast MRI. This central unenhancing portion coincides with the radiographic and clinical definition of necrosis among GBM patients. Moreover, as necrosis evolves, two relatively more contrast-enhancing rims are observed in relationship to the solid enhancing tumor surrounding the central necrosis in the later stages. The outer more prominently enhancing rim at the tumor border probably represents the infiltrating tumor edge, and the inner enhancing rim at the peri-necrotic region may represent locally infiltrating immune cells. The associated inflammation at the peri-necrotic region was further confirmed by immunohistochemical study of the temporal development of tumor necrosis. Neutrophils appear to be the predominant immune cell population in this region as necrosis evolves. This study shows central, brightly enhancing areas associated with inflammation in the tumor microenvironment may represent an early indication of necrosis development in GBM.

Abstract 6004: Surveying surface antigen expression in multiple myeloma preclinical models

Abstract Multiple myeloma (MM) is a progressive hematological cancer with a 5-year survival rate of 53% (1). Novel therapeutic strategies are being developed to target specific MM surface antigens. Yet, changes in antigen expression through MM progression are poorly understood in the clinic and have not been well characterized in preclinical models. Here, we were interested in understanding how BCMA, CD38, CD138 and HLA-DR surface expression patterns may be affected as MM progresses in preclinical models. To do so, ARH-77 and RPMI-8226 cells were inoculated subcutaneously while MM.1S, MM.1R, U266 and NCI-H929 cells were dosed intravenously into NSG mice. Tumor xenograft or bone marrow, whole blood and spleen were processed for flow cytometry analysis. Antigen expression was measured at different stages of tumor progression as well as in vitro. Soluble BCMA levels were assessed in the mouse serum by ELISA. ARH-77 and RPMI-8226 subcutaneous xenografts show preferential growth when inoculated in PBS and 50% growth factor-reduced Matrigel, respectively. BCMA expression was reduced in both ARH-77 and RPMI-8226 xenografts compared to in vitro culture. CD38 and HLA-DR expression increased as ARH-77/PBS and RPMI-8226/Matrigel tumor volume progressed. All 4 antigens were detected in the bone marrow from MM.1S, MM.1R and U266-bearing mice, as early as 5-6 weeks post intravenous injection. In contrast, antigens were not detected in any tissue from mice injected with NCI-H929, likely indicating failed engraftment. At 5 weeks, antigens were detected in the whole blood and spleen in MM.1S-bearing mice only, suggesting organ invasion and a more advanced disease-stage compared to MM.1R or U266. Experiments are being carried out to test antigen expression in tissue at late-stage tumor progression as well as serum BCMA levels in the intravenous MM models. These data highlight antigen expression differences in MM cells when analyzed in mouse tissue compared to in vitro culture. Like the widely variable expression observed between patients (2, 3), BCMA and CD138 were differentially expressed in the mouse bone marrow between models. Our observations suggest that commonly targeted antigens in MM vary kinetically in vivo and can be measured by flow cytometry. The present findings also support the use of RPMI-8226 and MM.1S cell lines when screening for antigen-specific immunotherapies and combinatorial studies for MM treatment.

Tumor-associated macrophages found at early tumor sites exhibit altered metabolism and contribute to tumor progression

Abstract Previously, we reported that macrophages at early tumor sites, before the tumors are even palpable, have a mixed phenotype with production of both pro- and anti-inflammatory cytokines, decreased production of reactive oxygen species, and increased dependence on oxidative phosphorylation. Here we show that depleting these tumor associated macrophages (TAM) impacted subsequent growth of the EMT6 tumor and metastasis of the 4T1 tumor supporting the contention that macrophages found at early tumor sites contribute to later stages of tumor progression. Since these TAM could not be clearly defined as M1 or M2 we set out to characterize them further focusing on their metabolism. We found that TAM present at early sites of 168, EMT6 and 4T1 tumors had elevated expression of pkm2, glut1 and ampk, and transwell assays revealed that these tumors could induce expression of pkm2, glut1 and ampk in bone marrow derived macrophages. Since tumor-derived lactate is known to induce alterations in white blood cells we investigated lactate production by these tumors and whether lactate could induce expression of these same genes. The data revealed that the 168, EMT6 and 4T1 tumors produced significant amounts of lactate and that lactate induced expression of pkm2, glut1 and ampk in bone marrow derived macrophages. Finally, we found that targeting lactate dehydrogenase or eIF2 at early tumor sites impacted growth of the EMT6 tumor, and targeting AMPK or GCN2 impacted metastasis of the 4T1 tumor. Collectively, these data support the contention that alterations in macrophages are already present at the early stages of tumor growth, before a tumor is even palpable, and that targeting metabolism at these early tumor sites can impact later stages of tumor progression.

Prognostic Potential of a PSAT1‐Associated Gene Signature in Identifying High‐Risk Patients in Early‐Stage EGFR‐Mutant Lung Cancer

De novocellular production of serine may contribute to tumor growth by providing precursors for macromolecular production and one‐carbon metabolism. Accordingly, multiple cancers exhibit increased expression of serine biosynthetic enzymes. For example, elevated PSAT1 (phosphoserine aminotransferase 1), which catalyzes the second step in converting 3‐phosphohydroxypyruvate to phosphoserine, is associated with poorer clinical outcomes. Several reports have implicated PSAT1 in the proliferation, migration, invasion, and chemo‐resistance of malignant cells. Our previous study found that PSAT1 translocated into the nucleus within EGFR‐activated NSCLC cells and was associated with poorer clinical outcomes of patients with EGFR‐mutant lung cancer, suggesting a relevant function for PSAT1 in EGFR‐driven tumorigenesis. In this study, we designed a bioinformatic approach to identify a PSAT1‐associated gene signature for EGFR‐mutant lung cancer (Fig. 1). Since PC9 cells are frequently utilized as an EGFR‐mutant lung cancer model, we speculated that a subset of PSAT1‐regulated genes independently contributes to EGFR‐driven lung tumorigenesis. For this, we transcriptionally profiled PSAT1 silenced PC9 cells. Analysis of the RNASeq identified 490 genes (279‐down, 211‐up) that were differentially expressed upon PSAT1 silencing (PSAT1‐mediated DEG). We next performed a comparative analysis between PSAT1‐mediated DEG and publicly available microarray datasets obtained from EGFR‐mutant patient tumors. Twenty‐five genes (13‐down, 12‐up) were differentially expressed in all EGFR‐mutant lung cancer datasets. These were classified as a PSAT1‐associated gene signature for EGFR‐mutant lung tumors and used to assess the relationship between this gene signature and both relapse‐free (RFS) and overall survival (OS) by the survival prediction tool from BRB‐ArrayTools. Thirteen and seventeen genes from the 25‐gene signature were found to predict the OS and RFS rates of EGFR‐mutant lung cancer, respectively. However, this signature failed to predict patient outcomes for either wild‐type or KRAS‐mutant lung tumors. Furthermore, the expression profile of RFS high‐risk patients with stage I EGFR‐mutant tumor phenocopies the high‐risk stage II tumor, supporting the prognostic power of PSAT1‐mediated gene signature for these EGFR‐mutant tumors. As early‐stage cancers dominate the gene signature obtained from human EGFR‐mutant lung tumors, analysis of genes involved in late‐stage tumor progression and metastasis is limited. Therefore, differentially expressed genes between parental PC9‐P and brain‐metastatic PC9‐BrM3 were extracted from the Nguyen et al.study (2009). Comparative analysis with RNA‐seq data found a potential PSAT1‐associated metastatic gene signature. In summary, our genomic approach yielded a PSAT1‐associated gene signature with prognostic value in EGFR‐mutant NSCLC patient outcomes.

329 Longitudinal immune profiling reveals unique myeloid and T cell phenotypes associated with spontaneous immunoediting in a novel prostate tumor model

OBJECTIVES/GOALS: Few preclinical models exist to study how tumors transition from prolonged stable disease ('equilibrium') to progressive disease ('escape'). We characterized a new murine tumor model the exhibits such behavior, and sought to identify and validate the role of unique tumor-infiltrating immune cell subsets in this process. METHODS/STUDY POPULATION: We evaluated growth of NPK-C1 (originally LM7304; received from Dr. Cory Abate-Shen at Columbia University), a cell line developed from spontaneous prostate cancer lung metastases in NPK mice (Nkx3.1CreERT2/+Ptenflox/floxKrasLSL-G12D/+R26R-LSL-YFP/+), in immune competent (C57BL/6) and immune deficient mice (J/Nu). We determined the role of CD4 and CD8 T cells in regulating the 'equilibrium to escape’ growth dynamics of NPK-C1 via in vivo cell depletions at key inflection points of tumor growth. To deeply profile the immune contexture of NPK-C1 at these inflection points, we developed a 28-color immunophenotyping panel for use on a Cytek Aurora spectral flow cytometer. We performed dimensionality reduction and clustering analyses on these data using tSNE and FlowSOM algorithms within FlowJo (v10.6). RESULTS/ANTICIPATED RESULTS: We found that activated CD4 effector T cells are enriched in regressing NPK-C1 tumors, highlighting a role for CD4 T cells in antitumor immunity. CD8 T cells are also important for NPK-C1 control; specifically central memory-like cytotoxic CD8 T cells. Depletion of either CD4 or CD8 T cells during the equilibrium phase of NPK-C1 growth confirmed the role of these cells in antagonizing NPK-C1 escape. Tregs as a whole were counterintuitively enriched in regressing tumors, however high dimensional analysis reveals their significant phenotypic diversity, with a number of Treg subpopulations enriched in progressing tumors. In the myeloid compartment, we found that iNOS+ DC-like cells are enriched in regressing tumors, while CD103+ DCs are associated with late stage tumor progression. DISCUSSION/SIGNIFICANCE: In total, these analyses of the NPK-C1 model provide novel insights into the roles of lymphoid and myeloid populations throughout key phases of tumor/immune co-evolution, and highlight a role for multi-dimensional flow cytometry-based analyses to more deeply understand immune cell dynamics in the tumor microenvironment.

Genome instability is associated with ethnic differences between Asians and Europeans in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the deadliest cancer types with diverse etiological factors across the world. Although large scale genomic studies have been conducted in different countries, integrative analysis of HCC genomes and ethnic comparison across cohorts are lacking.Methods: We first integrated genomes of 1,349 HCC patients from five large cohorts across the world and applied multiple statistical methods in identifying driver genes. Subsequently, we systematically compared HCC genomes and transcriptomes between Asians and Europeans using the TCGA cohort.Results: We identified 29 novel candidate driver genes, many of which are infrequent tumor suppressors driving late-stage tumor progression. When we systematically compared ethnic differences in the genomic landscape between Asian and European HCCs using the TCGA cohort (n = 348), we found little differences in driver frequencies. Through multi-modal integrative analysis, we found higher genomic instability in Asians together with a collection of molecular events ranging from tumor mutation burden (TMB), copy number alterations as well as transcriptomic subtypes segregating distinctively between two ethnic backgrounds. Strikingly, we identified an Asian specific transcriptomic subtype with multiple ethnically enriched genomic alterations, in particular chromosome 16 deletion, leading to a clinically aggressive RNA subgroup unique to Asians. Integrating multi-modal information, we found that survival models predict patient prognosis much better in Asians than in Europeans, demonstrating a higher potential for precision medicine applications in Asia.Conclusion: For the first time, we have uncovered an unprecedented amount of genomic differences segregating distinctively across ethnicities in HCC and highlighted the importance of differential disease biology and management in HCC across ethnic backgrounds.

Longitudinal Immune Profiling Reveals Unique Myeloid and T-cell Phenotypes Associated with Spontaneous Immunoediting in a Prostate Tumor Model.

The theory of cancer immunoediting, which describes the dynamic interactions between tumors and host immune cells that shape the character of each compartment, is foundational for understanding cancer immunotherapy. Few models exist that facilitate in-depth study of each of the three canonical phases of immunoediting: elimination, equilibrium, and escape. Here, we utilized NPK-C1, a transplantable prostate tumor model that we found recapitulated the three phases of immunoediting spontaneously in immunocompetent animals. Given that a significant portion of NPK-C1 tumors reliably progressed to the escape phase, we were able to delineate cell types and mechanisms differentially prevalent in equilibrium versus escape phases. Using high-dimensional flow cytometry, we found that activated CD4+ effector T cells were enriched in regressing tumors, highlighting a role for CD4+ T cells in antitumor immunity. CD8+ T cells were also important for NPK-C1 control, specifically, central memory-like cytotoxic CD8+ T cells. Regulatory T cells (Treg), as a whole, were counterintuitively enriched in regressing tumors; however, high-dimensional analysis revealed their significant phenotypic diversity, with a number of Treg subpopulations enriched in progressing tumors. In the myeloid compartment, we found that iNOS+ dendritic cell (DC)-like cells are enriched in regressing tumors, whereas CD103+ DCs were associated with late-stage tumor progression. In total, these analyses of the NPK-C1 model provide novel insights into the roles of lymphoid and myeloid populations throughout the cancer immunoediting process and highlight a role for multidimensional, flow-based analyses to more deeply understand immune cell dynamics in the tumor microenvironment.

Differential Impact of Calcitriol and Its Analogs on Tumor Stroma in Young and Aged Ovariectomized Mice Bearing 4T1 Mammary Gland Cancer.

(1) Background: Vitamin D compounds (VDC) are extensively studied in the field of anticancer properties, including breast cancer. Previously, we showed that calcitriol and its analogs (PRI-2191 and PRI-2205) stimulate metastasis in 4T1 murine mammary gland cancer models in young mice, whereas the reverse effect was observed in aged ovariectomized (OVX) mice; (2) Methods: We determined the phenotype of monocytes/macrophages using FACS and examined the expression of selected genes and proteins by Real-Time PCR and ELISA; (3) Results: Activities of VDC are accompanied by an increase in the percentage of Ly6Clow anti-inflammatory monocytes in the spleen of young and a decrease in aged OVX mice. Treatment of young mice with VDC resulted in an increase of CCL2 plasma and tumor concentration and Arg1 in tumor. In later stage of tumor progression the expression of genes related to metastasis in lung tissue was decreased or increased, in old OVX or young mice, respectively; (4) Conclusions: Pro- or anti-metastatic effects of calcitriol and its analogs in young or aged OVX mice, respectively, can be attributed to the differences in the effects of VDC on the tumor microenvironment, as a consequence of differences in the immunity status of young and aged mice.

A Dual Role of Type I Interferons in Antitumor Immunity.

Type I interferons (IFN-Is) are a family of cytokines that exert direct antiviral effects and regulate innate and adaptive immune responses through direct and indirect mechanisms. It is generally believed that IFN-Is repress tumor development via restricting tumor proliferation and inducing antitumor immune responses. However, recent emerging evidence suggests that IFN-Is play a dual role in antitumor immunity. That is, in the early stage of tumorigenesis, IFN-Is promote the antitumor immune response by enhancing antigen presentation in antigen-presenting cells and activating CD8+ T cells. However, in the late stage of tumor progression, persistent expression of IFN-Is induces the expression of immunosuppressive factors (PD-L1, IDO, and IL-10) on the surface of dendritic cells and other bone marrow cells and inhibits their antitumor immunity. This review outlines these dual functions of IFN-Is in antitumor immunity and elucidates the involved mechanisms, as well as their applications in tumor therapy.

Abstract A92: Analysis of lung-resident macrophages as potential regulators of disseminated tumor cell fate

Abstract The majority of cancer-related deaths are due to metastasis at secondary organs originating from solitary disseminated tumor cells (DTCs), which can remain dormant for several years. In the lung, some DTCs undergo a period of dormancy before proliferation to form metastasis, while others will not grow into metastatic lesions. As macrophages are key immune components of the microenvironment at distant sites, we aim to understand their potential role in regulating DTC dormancy and awakening. To test the contribution of lung macrophages to cancer cell proliferation, we first compared the effect of lung-resident alveolar macrophages (AMs) and bone marrow-derived monocytes (BM MOs) on cancer cell phenotypes. Our results indicate that AMs may suppress growth and induce EMT (CDH1lo/TWIST1hi phenotype) in cancer cells (of both early and late stages), while BM MOs promote growth. We further assessed the effect of these distinct macrophage populations on survival and proliferation of single cancer cells in a 3D matrigel assay to mimic single DTCs in the lung. Here, AMs appear to retain cancer cells as single cells, while BM MOs favor a switch to proliferation and colony growth. We further used conditioned media (CM) from lung cultures from naive and macrophage-depleted animals to study how organ-specific cues from macrophages affected tumor cell growth. We performed a proteomic analysis of the lung secretome and identified DKK3 as differentially produced before and after macrophage depletion. DKK3, a regulator of Wnt signaling, was absent in the depleted condition. We then tested its effect on cell proliferation in mammary cancer cell sphere formation assays, and we observed that DKK3 increased sphere formation and growth of early- and late-stage cells, respectively. We will next test whether DKK3 production is derived from AMs, BM MOs, or other lung resident cells and we will profile AMs and BM MOs from early and late stages of tumor progression to determine their gene expression profiles at different times of DTC colonization. To explore the role of AMs in dormancy in vivo, we will perform AM depletion in the CD169-DTR animal model. Here, we will deplete specifically tissue-resident AMs and determine DTC fate after tail-vein injection of breast cancer cells. Our study will thus allow us to determine the contribution of different macrophage populations to metastatic dormancy. Citation Format: Erica Dalla, Maria Casanova-Acebes, Margaret Hung, Nina Linde, Miriam Merad, Julio Aguirre-Ghiso. Analysis of lung-resident macrophages as potential regulators of disseminated tumor cell fate [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A92.

Microenvironment-Dependent Gradient of CTL Exhaustion in the AE17sOVA Murine Mesothelioma Tumor Model.

The immune system, and in particular, cytotoxic CD8+ T cells (CTLs), plays a vital part in the prevention and elimination of tumors. In many patients, however, CTL-mediated tumor killing ultimately fails in the clearance of cancer cells resulting in disease progression, in large part due to the progression of effector CTL into exhausted CTL. While there have been major breakthroughs in the development of CTL-mediated “reinvigoration”-driven immunotherapies such as checkpoint blockade therapy, there remains a need to better understand the drivers behind the development of T cell exhaustion. Our study highlights the unique differences in T cell exhaustion development in tumor-specific CTL which arises over time in a mouse model of mesothelioma. Importantly, we also show that peripheral tumor-specific T cells have a unique expression profile compared to exhausted tumor-infiltrating CTL at a late-stage of tumor progression in mice. Together, these data suggest that greater emphasis should be placed on understanding contributions of individual microenvironments in the development of T cell exhaustion.