Immune Suppression Gene Research Articles

Single-cell RNA sequencing highlights the role of proinflammatory fibroblasts, vascular endothelial cells, and immune cells in the keloid immune microenvironment.

Keloids, characterized by an aberrant wound-healing process and a highly complex immune microenvironment, pose significant challenges for clinical management. Fibroblasts and vascular endothelial cells (VEC) were identified as the leading cells of keloid development. However, their roles in the keloid immune landscape have yet to be thoroughly elucidated. To explore the functional state of cells in the immune landscape of keloids, we conducted a single-cell RNA sequencing analysis on the tissue from three keloid lesions and two specimens of healthy skin. We simultaneously utilized available keloid data from the public database for external validation. Specific subsets, such as proinflammatory fibroblasts (piF) and VEC, were markedly elevated in lesional skincompared to normal skin. Subsequent differential gene expression and Gene Ontology analyses indicated that these subsets may be involved in shaping the microenvironment. In keloids, there is an increased expression of immune-associated genes (P < 0.05), including TNFRSF6B, HGF, and TGFB3, alongside a decreased expression of inflammatory chemokines in the piF. Moreover, the significant upregulation of immune suppressive genes (P < 0.05), including CD39, CD73, and HIF1A, suggested the potential involvement of VEC as a conditional immune subpopulation in the keloid microenvironment. Immune cell communication analysis revealed preferential enrichment of macrophages and Tregs, highlighting intensified macrophage-centered interactions within the keloid microenvironment. Our study highlighted the role of piF and VEC in the immune microenvironment of keloids for the first time, providing potential targets for therapeutic development.

Exploring markers of immunoresponsiveness in papillary thyroid carcinoma and future treatment strategies

BackgroundThe study summarizes the potential use of immunotherapy for BRAF-mutated papillary thyroid cancer (PTC) by analyzing the immune profile of City of Hope PTC patient samples and comparing them to...

Staphyloccocus aureus biofilm, in absence of planktonic bacteria, produces factors that activate counterbalancing inflammatory and immune-suppressive genes in human monocytes.

Staphyloccocus aureus (S. aureus) is a major bacterial pathogen in orthopedic periprosthetic joint infection (PJI). S. aureus forms biofilms that promote persistent infection by shielding bacteria from immune cells and inducing an antibiotic-tolerant metabolic state. We developed an in vitro system to study S. aureus biofilm interactions with primary human monocytes in the absence of planktonic bacteria. In line with previous in vivo data, S. aureus biofilm induced expression of inflammatory genes such as TNF and IL1B, and their anti-inflammatory counter-regulator IL10. S. aureus biofilm also activated expression of PD-1 ligands, and IL-1RA, molecules that have the potential to suppress T cell function or differentiation of protective Th17 cells. Gene induction did not require monocyte:biofilm contact and was mediated by a soluble factor(s) produced by biofilm-encased bacteria that was heat resistant and >3 kD in size. Activation of suppressive genes by biofilm was sensitive to suppression by Jak kinase inhibition. These results support an evolving paradigm that biofilm plays an active role in modulating immune responses, and suggest this occurs via production of a soluble vita-pathogen-associated molecular pattern, a molecule that signals microbial viability. Induction of T cell suppressive genes by S. aureus biofilm provides insights into mechanisms that can suppress T cell immunity in PJI.

Abstract P57: A Transcriptome-Wide Meta-Analysis Reveals Lack Of Cancer-Cell Intrinsic Determinants Of Response To Immune Checkpoint Blockade

Abstract Immune-checkpoint blockade therapy (ICB) has conferred significant and durable clinical benefit to some cancer patients. However, most patients do not respond to ICB, and reliable biomarkers of ICB response are needed to improve patient stratification. Current FDA-approved biomarkers for ICB include PD-L1 expression, microsatellite instability (MSI), and high tumor mutation burden (TMB) of &amp;gt;10mutations/Mb. In addition, gene expression signatures of tumor immune phenotypes have been proposed to predict for ICB response. Despite these efforts, there is a lack of large-scale transcriptome-wide studies to identify and characterize potential biomarkers of ICB response conserved across cancer types in a cell type specific manner. Here, we performed a transcriptome-wide meta-analysis across 1,486 tumors from ICB-treated patients and tumors with expected ICB outcomes based on microsatellite status. Using a robust transcriptome deconvolution approach, we inferred cancer and stroma-specific gene expression differences and identified cell-type specific features of ICB response across cancer types. We identified 59 stroma-specific differentially expressed genes associated with ICB-response, which were strongly enriched in immune-related pathways and processes. Stromal expression of CXCL9, CXCL13, IFNG, and CD274 were among the top positive determinants of ICB response, consistent with current knowledge. Interestingly, we also identified a group of potential immune-suppressive genes, including FCER1A, associated with poor response to ICB. Surprisingly, the unbiased transcriptome-wide analysis failed to identify cancer-cell intrinsic features of ICB response conserved across tumor types. Overall, our results suggest that cancer cells lack tissue-agnostic molecular determinants of ICB response, which has implications for the development of improved ICB diagnostics and treatments. Citation Format: Yu Amanda Guo, Tanmay Kulshrestha, Mei Mei Chang, Irfahan Kassam, Egor Revkov, Simone Rizzetto, Aaron C. Tan, Daniel S.W. Tan, Iain Beehuat Tan, Anders Jacobsen Skanderup. A Transcriptome-Wide Meta-Analysis Reveals Lack Of Cancer-Cell Intrinsic Determinants Of Response To Immune Checkpoint Blockade [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P57.

Abstract 4402: A genomic insertion/deletion variant activates immune escape and tumor promoting programs in breast cancer

Abstract Purpose: Despite all the knowledge we have concerning cancer cells’ immunesuppressive ability, we still don’t understand whether the mechanisms upregulatingimmune suppression in cancer cells are patient specific. This information is critical forthe generation of tailored treatments that recruit the immune system for the eradicationof cancer disease. In the past, we showed that super-enhancers, composed of cluster ofenhancers spanning over long genomic regions characterized by having potent generegulatory activity, are critical for the upregulation of immune suppressive genes,including CD47, within the tumor. Recently we found a DNA insertion/deletion variant (8bp in size) within a super-enhancer located between the CD47 and Linc00636 genes,that is present more often in European (53%) than Asian (12%) populations and whichassociates with breast cancer. Moreover, sequence analyses of patient-derived breasttumors (44) and gynecological tumor samples (23) demonstrated a significantassociation of this insert with breast cancer and not with gynecological cancer. Thus, wehypothesize that the insertion variant regulates genes that drive immune escape andbreast cancer specifically in patient populations that carry the insert version. Methods: To test this, we CRISPR deleted the variant’s insertion and CRISPR-VP64induced its activation and quantified its effect on target genes and encoded proteinsusing high throughput RNA sequencing and immunofluorescence flow cytometryanalyses respectively. Results: CRISPR-VP64 induction of the 8bp insertion confirmed that the insertion issufficient to regulate gene expression of CD47 and of an anti-tumor, long intergenicnon-coding RNA (Linc00636) in breast cancer cells. Furthermore, we observed thatCRISPR deletion of the 8bp insertion increases CD47 levels, while dramaticallyincreasing expression of Linc00636 and reducing the expression of CD44, CXCL14,MUC1 genes involved in invasion and migration. Our work shows that the insertion ofan 8bp is responsible for stabilizing upregulation of CD47 (an immune suppressivesignal), downregulating Linc00636 (a modulator of tumor metastasis), and upregulating,as a result, the expression of cell surface receptors involved in metastasis andtumorigenesis. Conclusions: Here we found a genomic insert within a super-enhancer that unleashesthe upregulation of Linc00636 and of a program that promotes breast cancer, mostlikely in populations of patients carrying this variant. Our findings are important tounderstand patient specific genomic bifunctional modulators of immune suppressionand tumor progression for the design of more personalized treatments geared towardprecision medicine. Citation Format: Paola Betancur, Carolina DiBenedetto, Daniza Diane Acenas, Anthony Rodriguez, Alysia Thach. A genomic insertion/deletion variant activates immune escape and tumor promoting programs in breast 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 4402.

Abstract 5583: Helicobacter pylori induces the wee1, promoting immune suppressive in human gastric adenocarcinoma

Abstract Background: Gastric cancer (GC) is the fourth most common cause of cancer-related death worldwide. GC has a dismal 5-year survival rate of about 32%. Helicobacter pylori (H. pylori) infection is the strongest risk factor for gastric carcinogenesis. 75-90% of gastric cancer patients exhibit positive H. pylori serology. WEE1 is a nuclear kinase that regulates cell cycle progression by inhibiting cyclin-dependent kinases. Methods &amp; Results: Analysis of non-cancer normal stomach tissue (n=360) and human GC samples (n=1221) identified that WEE1 mRNA was significantly overexpressed in GC. Using immunohistochemistry (IHC) staining in 41 normal and 44 GC human samples, the data indicated that WEE1 protein was significantly overexpressed in GC. Western blot and immunofluorescence (IF) staining confirmed that H. pylori infection induces WEE1 overexpression in GC cells with an unexpected cytosolic localization of WEE1 in GC cells. Tff1 knockout GC mouse model (Tff1-/-) was infected with mouse-adapted H. pylori strain PMSS1. Western blot and immunohistochemistry data demonstrated that H. pylori infection induced WEE1 expression in Tff1 -/- HGD/cancer mouse gastric tissues compared to control Tff1 -/- mouse stomach. To study how H. pylori infection induces WEE1 in GC, analysis from miRTAR, Target Scan, and miRDB demonstrated that WEE1 is predicted to be targeted by miR-497-5p. Using qRT-PCR analysis, we assessed 30 normal and 30 GC human tissue samples, revealing a pronounced downregulation of miR-497-5p in GC specimens. Western blot analysis confirmed that the restoration of miR-497-5p in GC cells markedly reduced WEE1 protein expression. Interestingly, H. pylori infection further decreased miR-497-5p levels in GC cells, upregulating WEE1 protein expression. Since the miR-497-5P host gene promoter region contains CpG islands, we confirmed that treatment with 5 Azacytidine (demethylating agent) or DNMT1 knockdown induced expression of miR-497-5p with decreased WEE1 level in GC cells. Furthermore, our preliminary results indicate that aberrant WEE1 overexpression in GC demonstrates a significant positive correlation with immune-suppression gene signatures, underscoring its predictive value for unfavorable patient survival outcomes in GC. Using the TFF1 knockout mouse GC model, we found that WEE1 inhibition notably decreases the number and activation of CD4+ and CD8+ T cells in neoplastic gastric tissues. These findings also indicate a crucial relationship between WEE1 and the immunosuppressive microenvironment in GC, establishing WEE1 as a promising therapeutic target to enhance therapeutic efficacy and overcome immunotherapy resistance in GC. Conclusions: Our findings demonstrated a novel mechanism in which H. pylori activates the WEE1 in GC, promoting an immune suppressive microenvironment. WEE1 inhibition with immunotherapy could be a promising therapeutic method for GC patients. Citation Format: Md Obaidul Islam, Krishnapriya Thangaretnam, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, Wael El-Rifai, Zheng Chen. Helicobacter pylori induces the wee1, promoting immune suppressive in human gastric adenocarcinoma [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 5583.

IGSF8 is a potential target for the treatment of gliomas

BackgroundImmunoglobulin superfamily member 8, or IGSF8, is a member of the recently identified immunoglobulin family of proteins. It is mostly produced on cell membranes and has a unique transmembrane structure. It has recently been demonstrated that there is a strong correlation between the expression variation of IGSF8 and the growth of gliomas. Therefore, we used data from the TCGA and CGGA databases to evaluate the function of IGSF8. MethodsThe TCGA and GTEx data sets' RNA-seq data were utilized to examine IGSF8 expression. The Gene Cards database was utilized to get IGSF8 protein data. The Cluster Profiler data package was used to carry out the IGSF8 enrichment study. The GO and KEGG databases were used to examine the relationship between IGSF8 and cellular physiological and biochemical processes. The TCGA immune cell infiltration scores were obtained from online databases and published studies. Clinical survival data from TCGA and CGGA were used to investigate the predictive significance of IGSF8. ResultsTGGA revealed that the majority of cancers had differential expression of IGSF8. IGSF8 was discovered to be enriched in numerous significant pathways in tumor cells by GO and KEGG. Moreover, a strong correlation was seen between the expression of IGSF8 and the immunomodulatory interactions that occur between non-lymphocytes and lymphocytes. T-cell infiltration, immunological checkpoints, immune-activating and immune-suppressive genes, chemokines, and chemokine receptors were all strongly correlated with IGSF8 expression. Lastly, the TCGA and CGGA databases showed a strong correlation between IGSF8 and the grade and prognosis of gliomas. ConclusionAccording to our findings, IGSF8 may be a glioma marker. In order to control the immunological microenvironment, IGSF8 may cooperate with a number of immune checkpoints. This information may be utilized to create novel targeted immunotherapy medications.

Identification of SLC12A8 as a Valuable Prognostic Biomarker and Immunotherapeutic Target by Comprehensive Pan-Cancer Analysis

Solute carrier family 12 member 8 (SLC12A8) is a nicotinamide mononucleotide transporter. Despite emerging evidence supporting its potential involvement in oncogenesis, a systematic pan-cancer analysis of SLC12A8 has not been performed. Thus, this research aimed to explore the prognostic implications of SLC12A8 and assess its possible immune-related functions across 33 different tumor types. And multiple datasets were retrieved from the databases of TCGA, GTEx, Broad Institute CCLE, TISCH, HPA, and GDSC2. After this data acquisition, bioinformatics analyses were conducted to assess the potential involvement of SLC12A8 in cancer pathogenesis. These analyses focused on examining the relationship between SLC12A8 and prognosis, drug sensitivity, chemotherapy response, immune checkpoints (ICPs), immune cell infiltration, and immunotherapy efficacy across various tumor types. Furthermore, experimental methods such as EdU assay, wound healing assay, and transwell assay were conducted to evaluate the cell proliferative and invasive abilities. Finally, the data analysis demonstrated that SLC12A8 was differentially expressed and predicted unfavorable survival outcomes in the majority of the tumor types in the TCGA dataset. Furthermore, a notable upregulation in the expression of SLC12A8 mRNA and protein was observed in cancer tissues compared to normal tissues. Additionally, the SLC12A8 levels demonstrated a strong association with ICPs, chemokines, immune-activating genes, immune-suppressive genes, chemokine receptors, chemotherapy response, and immunotherapy efficacy. In vitro experiments substantiated that knockdown of SLC12A8 restricted the malignant phenotypes of MDA-MB-231 and BT-549 cells. So SLC12A8 holds promise as a cancer biomarker with the capacity to interact with other ICPs to synergistically regulate the immune microenvironment. Thus, the identification of SLC12A8 contributes to the development of novel therapeutic strategies for enhancing the efficacy of immunotherapy.

Leukemia Blasts Induce NK-like Dysfunction in AML-Specific T Cells

Introduction Efforts to enhance T cell-mediated acute myeloid leukemia (AML) eradication face challenges possibly due to unique mechanisms of leukemia-induced T-cell dysfunction compared to exhaustion observed in solid tumors(Penter et al., Blood 2023). We previously showed the efficacy of Wilms' Tumor (WT) Antigen 1-specific T-cell receptor (TCR C4) inserted into CD8 + T cells (T TCR-C4) in preventing AML recurrence after hematopoietic cell transplantation (HCT) (Chapuis et al., Nat Med 2019). This led us to investigate the safety and efficacy of T TCR-C4 in post-HCT relapsed patients. Methods In this Phase I/II study conducted at Fred Hutchinson Cancer Center, 15 patients were enrolled and prospectively monitored for disease response. We manufactured T TCR-C4 using a lentiviral transduction system (Chapuis et al, Nat Med 2019) and performed comprehensive analyses of bone marrow (BM) and peripheral blood (PB) samples using single-cell RNA sequencing (scRNAseq) and TCRseq. Results T TCR-C4 treatment showed no survival advantage compared to matched controls (n =17). We hypothesized that active AML exacerbates T-cell dysfunction compared to patients who received prophylactic T TCR-C4 treatment. Identifying tumor-specific T cells poses a challenge in scRNAseq. By utilizing the WT1-transgene (WT1tg) sequence, we built a custom reference genome to dissect the transcriptional state of leukemia-specific cells. In PB scRNAseq profiles of 4 patients (24,472 CD8 + T cells) with post-infusion persistent blasts and T TCR-C4, we identified 5 main CD8 + T-cell signatures: Naïve, Stem-like, Activated-Exhausted, WT1tg positive, and NK-like (Figure 1). The NK-like signature has been associated with dysfunction in CAR-T cells (Good et al., Cell 2021). T TCR-C4 cells exhibited an NK-like skewing, although the expression of these markers was not as high as in endogenous NK-like CD8 + T cells. Trajectory inference revealed that NK-like endogenous CD8 + T cells represented the terminal state of differentiation, while T TCR-C4, despite expressing some NK-like markers, were in an intermediate state of differentiation along the trajectory (Figure 2). Combining scTCRseq data with proliferation signatures (Zheng et al., Science 2021) we found that WT1tg exhibited clonal expansion and proliferation, suggesting a functional intermediate state. We validated the 5 CD8 + T-cell differentiation states in BM samples (7,719 CD8 + T cells) from our cohort and in a publicly available large-scale BM CD8 + T-cell scRNAseq dataset (68,545 CD8+ T cells), with NK-like as the last differentiation state along the trajectory. Differential gene expression (DGE) of CD8 + T cells between active AML (AML+) and blasts-not-detectable (AML-) timepoints revealed reduced expression of stem-like and increased expression of NK-like markers in AML+ vs. AML- suggesting that leukemia drives NK-like terminal differentiation in T TCR-C4. To investigate if the NK-like derangement is AML-specific or a general dysfunction mechanism, we performed DGE between T TCR-C4 vs. TCR-T cells mesothelin-specific (T MSLN). The analysis revealed overexpression of exhaustion markers (CTLA4, PDCD1, TOX, CXCR6, NR4A2) in T MSLN and NK-like markers (NKG7, KLRD1, KLRG1, S1PR5) in T TCR-C4. To explore leukemia immune-escape mechanisms, we studied blasts gene expression. One patient who initially responded to T TCR-C4, exhibited monocytic blast differentiation upon relapse. We found overexpression of immune suppression genes (IL10, TNF, reactive oxygen species) in monocytic blasts at relapse vs. blasts before T TCR-C4 infusion. Published AML scRNAseq confirmed NK-like skewing in monocytic leukemia patients. Ongoing in-vitro assays will help to dissect the mechanisms of T TCR-C4 dysfunction. Discussion Our study reveals a novel AML-specific NK-like signature of T-cell dysfunction, exacerbated when AML blasts are present, potentially contributing to the restricted efficacy observed in TCR-T cell therapies. We observed that monocytic blast differentiation and the subsequent formation of an immune-suppressive environment imply the existence of intricate immune escape mechanisms. This study provides a critical foundation for the development of innovative interventions aimed at circumventing the dysfunction observed in cell-based therapies and fostering long-term remission in AML patients.

HIC1 interacts with FOXP3 multi protein complex: Novel pleiotropic mechanisms to regulate human regulatory T cell differentiation and function

Transcriptional repressor, hypermethylated in cancer 1 (HIC1) participates in a range of important biological processes, such as tumor repression, immune suppression, embryonic development and epigenetic gene regulation. Further to these, we previously demonstrated that HIC1 provides a significant contribution to the function and development of regulatory T (Treg) cells. However, the mechanism by which it regulates these processes was not apparent. To address this question, we used affinity-purification mass spectrometry to characterize the HIC1 interactome in human Treg cells. Altogether 61 high-confidence interactors were identified, including IKZF3, which is a key transcription factor in the development of Treg cells. The biological processes associated with these interacting proteins include protein transport, mRNA processing, non-coding (ncRNA) transcription and RNA metabolism. The results revealed that HIC1 is part of a FOXP3-RUNX1-CBFB protein complex that regulates Treg signature genes thus improving our understanding of HIC1 function during early Treg cell differentiation.

One Step Nucleic Acid Amplification (OSNA) Lysate Samples Are Suitable to Establish a Transcriptional Metastatic Signature in Patients with Early Stage Hormone Receptors-Positive Breast Cancer.

The One Step Nucleic Acid Amplification (OSNA) is being adopted worldwide for sentinel lymph nodes (SLNs) staging in breast cancer (BC). As major disadvantage, OSNA precludes prognostic information based on structural evaluation of SLNs. Our aim is to identify biomarkers related to tumor-microenvironment interplay exploring gene expression data from the OSNA remaining lysate. This study included 32 patients with early stage hormone receptors-positive BC. Remaining OSNA lysates were prepared for targeted RNA-sequencing analysis. Identification of differentially expressed genes (DEGs) was performed by DESeq2 in R and data analysis in STATA. The results show that, in metastatic SLNs, several genes were upregulated: KRT7, VTCN1, CD44, GATA3, ALOX15B, RORC, NECTIN2, LRG1, CD276, FOXM1 and IGF1R. Hierarchical clustering analysis revealed three different clusters. The identified DEGs codify proteins mainly involved in cancer aggressiveness and with impact in immune response. The overexpression of the immune suppressive genes VTCN1 and CD276 may explain that no direct evidence of activation of immune response in metastatic SLNs was found. We show that OSNA results may be improved incorporating microenvironment-related biomarkers that may be useful in the future for prognosis stratification and immunotherapy selection. As OSNA assay is being implemented for SLNs staging in other cancers, this approach could also have a wider utility.

1700P Radiation therapy elicits a consistent myeloid and lymphoid immune response in breast and rectal cancer

Radiotherapy (RT) is broadly used for cancer treatment, including early breast (eBRCA) and rectal cancers (RC). However, many components of the tumor microenvironment (TME) contribute to RT resistance. RT induces cancer cell death resulting in tumor antigen release that triggers the recruitment of a variety of immune cells that can act either stimulatory or suppressive on anti-tumor immunity. A deeper understanding of the immune suppressive components of anti-tumor immunity is integral to discover novel treatments to improve RT effectiveness. In this study, RT induced transcriptional changes were identified that associate with cellular consequences that may promote radio-resistance. Moreover, the delineation of tumor-specific changes can help selecting patients undergoing RT that are likely to have tumor recurrence. Datasets of patients with eBRCA (Horton 2015) and RC (Petty 2009) that received single-dose RT were analyzed. Gene signatures of immune activation and suppression were summarized for each patient as an average of the standard score and evaluated at the baseline and after RT. Fractions of immune cell populations were estimated by cell type deconvolution using xCell (Aran 2017) and compared pre and post RT. Evaluation of gene signatures of immune activation (inflammatory response, T-cell inflammation) and immune suppression (M2 macrophages, TGF-b signaling) in eBRCA and RC patients showed a consistent increase in pre compared to post RT. The comparison of score fold-change (pre-post RT) showed a stronger increase of the signatures in RC compared to eBRCA, with similar change for the TGF-b signaling score. Cell type deconvolution analysis showed that M1 and M2 macrophages and cancer-associated fibroblasts increase after RT in both tumor types, while NK T cells decrease. The main differences were found in neutrophils and regulatory T cells that have opposite trends in the two tumor types. The results showed an overall increase of immune activation and suppression genes after RT, more pronounced in RC compared to eBRCA patients. Moreover, the TME of RC showed a more immune-suppressive profile with an increase of regulatory T cells in addition to M2 macrophages.

Single-Nucleus RNA Sequencing and Spatial Transcriptomics Reveal the Immunological Microenvironment of Cervical Squamous Cell Carcinoma.

The effective treatment of advanced cervical cancer remains challenging. Herein, single‐nucleus RNA sequencing (snRNA‐seq) and SpaTial enhanced resolution omics‐sequencing (Stereo‐seq) are used to investigate the immunological microenvironment of cervical squamous cell carcinoma (CSCC). The expression levels of most immune suppressive genes in the tumor and inflammation areas of CSCC are not significantly higher than those in the non‐cancer samples, except for LGALS9 and IDO1. Stronger signals of CD56+ NK cells and immature dendritic cells are found in the hypermetabolic tumor areas, whereas more eosinophils, immature B cells, and Treg cells are found in the hypometabolic tumor areas. Moreover, a cluster of pro‐tumorigenic cancer‐associated myofibroblasts (myCAFs) are identified. The myCAFs may support the growth and metastasis of tumors by inhibiting lymphocyte infiltration and remodeling of the tumor extracellular matrix. Furthermore, these myCAFs are associated with poorer survival probability in patients with CSCC, predict resistance to immunotherapy, and might be present in a small fraction (< 30%) of patients with advanced cancer. Immunohistochemistry and multiplex immunofluorescence staining are conducted to validate the spatial distribution and potential function of myCAFs. Collectively, these findings enhance the understanding of the immunological microenvironment of CSCC and shed light on the treatment of advanced CSCC.

Abstract 2548: The central nervous system immune cell interactome is a function of cancer lineage, tumor microenvironment and STAT3 expression

Abstract Introduction: Deconstructive immune cell profiling of central nervous system (CNS) tumors has focused on the tumor, excluding interrogation of the tumor microenvironment (TME). Integrated spatial analysis can ascertain the cell interactome and may be a key biomarker for effective anti-tumor immune responses. Methods: En bloc resections of glioma (n=10) and lung metastasis (n=10) to preserve the tissue architecture, underwent tissue segmentation and high dimension opal 7-color multiplex imaging. Bioinformatic analysis of scRNA was used to infer immune cell functionality. Results: CD3+ T cell frequency was equivalent between CNS cancer lineages. Within gliomas T cells were confined to the perivascular space and the infiltrating edge. In lung metastasis, T cells are confined to the tumor stroma. CD163+ macrophages predominate in brain metastasis throughout the TME (p&amp;lt;0.05), while CD68+ monocytes (CD68+, CD11c+CD68+, and CD11+CD68+CD163+) are more common in gliomas (p&amp;lt;0.05). T cell dyad and cluster immune interactions were more common in the absence of nuclear STAT3 expression. T cells usually interact with CD163+ macrophages as dyads in metastasis at the brain interface (p=0.031) and within tumor (p=0.0009); in clusters throughout the TME (interface: p=0.024; tumor: p=0.01; necrosis: p=0.045), and as STAT3+ dyads and cluster interactions in the tumor (p&amp;lt;0.05). Immune suppressed CD11c+CD163+ dendritic cells (tumor: p=0.036; and necrosis p=0.020) predominate in metastasis. In contrast, gliomas typically lacked dyad and cluster interactions except for T cell and CD68+ dyads in the tumor (p=0.023). Bioinformatic analysis of CD45+ scRNA seq data revealed that the majority of innate immune populations express both pro-inflammatory and immune suppressive genes and that subsets of CD68+ and CD11c+CD68+ cells expressed markers such as TMEM119, P2YR13 and CX3CR1 that identify microglia. Conclusion: Current therapies are targeted to cell populations and singular pathways. Immunosuppressive macrophages dominate within the TME and targeting this population may create an environment that favors T cell activation and effective immune responses. Furthermore, the immune interactome, an important event for anti-tumor immune response, is a function of cancer lineage, TME, and STAT3 expression, which will gain relevance for future therapeutics directed to modulating these interactions. Citation Format: Hinda Najem, Martina Ott, Cynthia Kassab, Arvind Rao, Ganesh Rao, Anantha Marisetty, Adam M. Sonabend, Craig Horbinski, Roel Verhaak, Anand Shakar, Santhoshi Krishnan, Frederick S. Varn, Victor A. Arietta, Pravesh Gupta, Sherise D. Ferguson, Jason Huse, Gregory N. Fuller, James Long, Dan Winskowski, Ben Freiberg, C. David James, Leonidas C. Platanias, Maciej S. Lesniak, Jared K. Burks, Amy B. Heimberger. The central nervous system immune cell interactome is a function of cancer lineage, tumor microenvironment and STAT3 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2548.

Abstract 5641: Tumor cell intrinsic factors in colorectal cancer immune evasion

Abstract The poor response of most colorectal cancer (CRC) patients to immunotherapy is a major unmet clinical need. Although early detection through screening has reduced CRC incidence, the prognosis for metastatic CRC is poor. In the past decade, immune checkpoint inhibitors (ICIs) have been a major clinical breakthrough with promising clinical benefits in many cancer types, but its efficacy is limited in most (~85%) CRC patients. ICIs preferentially have robust responses from tumors with pre-existing T cell infiltration. However, most CRC lacks robust T-cell infiltration due to immune evasion mechanisms. Therefore, it is crucial to characterize factors shaping immune evasion to improve the response to ICIs. We hypothesize that targeting tumor-cell intrinsic factors can overcome T cell exclusion and will sensitize the tumor to ICIs. To elucidate the factors underlying ICI resistance, it is critical that preclinical models faithfully recapitulate the tumor physiology and genetics of the human disease. Therefore, we developed a tumor organoid (tumoroids) model system adapted from Jacks lab that harbors shApc, KrasG12D, p53-/- and Smad4-/- (shAKPS) mutations, which are co-mutated with high frequency and associated with poor prognosis. We demonstrated that tumoroids with only shApc and KrasG12D mutations can form a solid tumor in two weeks. Addition of p53-/- and Smad4-/- mutations drives invasiveness and metastasis. The shAKPS tumoroids present key features of advanced CRC that are unresponsive to ICIs including invasive tumor, metastasis, and T cell exclusion. Using CRISPR/Cas9 screening and integrative immune score analysis, we identified potential immune evasive genes. To define their expression profile in the tumor, we performed TCGA data analysis in fourteen different cancer types. Furthermore, we performed correlation tests to determine their role in immune cell infiltration and T cell dysfunction. Our results show that these immune suppressive genes are overexpressed in tumor tissues, and negatively correlate with CD8+ T cell infiltration. Our findings suggest that overexpression of immune suppressive genes in cancer cells may cause T cell dysfunction and exclusion in tumors. Furthermore, the functional role of these immune suppressive genes will be characterized using shAKPS mutant organoids and in vivo orthotopic CRC tumor models. Citation Format: Dechen Wangmo, Xianda Zhao, Angelo Yuan, Travis Gates, Subree Subramanian. Tumor cell intrinsic factors in colorectal cancer immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5641.

FAM46C as a Potential Marker for Pan-Cancer Prognosis and Predicting Immunotherapeutic Efficacy.

Background: FAM46C is a common mutated gene in tumours. A comprehensive understanding of the relationship between FAM46C expression and pan-cancer can guide clinical prognosis and broaden the immunotherapeutic targets. Methods: Data from The Cancer Genome Atlas and Genotype-Tissue Expression (GTEx) databases were obtained, and gene expression of different tumour types and stages was analysed. Immunohistochemical analysis was performed to detect differences in the FAM46C protein levels in normal and cancerous tissues. The genetic variation of FAM46C was characterised using cBioPortal. The clinical prognostic value of FAM46C and the impact of FAM46C expression levels on the prognosis of patients with different types of cancer were assessed based on Kaplan–Meier and Cox regression analyses. Gene set enrichment analysis (GSEA) was used to analyse the pathways associated with FAM46C. Correlations between FAM46C expression levels and immune infiltration were assessed using the TIMER2 database and CIBERSORT algorithm, and correlations between FAM46C expression and the ESTIMATE, immune and stromal scores were analysed using the ESTIMATE algorithm. In addition, we also analysed the correlation between FAM46C expression and immune activation, suppression genes and immune chemokines. Results: The expression level of FAM46C was correlated with the prognosis of most tumours, and low expression levels often suggested a poor prognosis. FAM46C was positively correlated with the abundance of CD4+ T cells, CD8+ T cells and plasma B lymphocytes in the tumour microenvironment. FAM46C exhibited a strong correlation with immunomodulatory pathways, immunomodulatory factors and immune markers. In addition, high FAM46C expression correlated with tumour mutational burden in acute myeloid leukaemia and microsatellite instability in endometrial cancer. Conclusion: Our study suggests that FAM46C can be a potential prognostic marker for pan-cancer, is closely associated with immune regulation and may be an immune checkpoint to guide future clinical immunotherapy.

Pan-Cancer Analysis Predicts FOXS1 as a Key Target in Prognosis and Tumor Immunotherapy.

PurposeOnly a few studies have reported the role of FOXS1, a transcriptional factor, in the tumor development process. In this article, we investigate the function of FOXS1 in distinct neoplastic development and the tumor immune microenvironment (TIME).Patients and MethodsThe latent roles of FOXS1 in various tumors were prospected based on TCGA, GTEx, CCLE, GEPIA2, cBioPortal, TIMER, ImmuCellAI databases, GSVA datasets, GSEA datasets, and R packages. The expression difference, gene alteration, clinical characteristics, prognostic values, biological mechanism, potential pathways, tumor microenvironment, and immune cell infiltration related to FOXS1 were appraised.ResultsFOXS1 was strongly expressed in pan-cancer, and this gene was associated with low survival rates. FOXS1 was linked to many pathways that are cancer-promoting and immune-related. The expression of this transcriptional factor in cancers was positively related to immune cell infiltration, especially M2-like macrophages and Treg cells. In addition to that, FOXS1 demonstrated a positive relationship with many immune-suppression genes, such as TGFB1 and ARORA2A.ConclusionOur study identified an oncogenic effect of FOXS1, which may play a vital role as a prognosticative biological marker in pan-cancer. Exorbitant expression of FOXS1 is associated with high TAMs and Treg cells infiltration. These cells have an immunosuppressive function and promote the development of the immunosuppressive tumor microenvironment. The research of FOXS1 provided a potential drug target for tumor immunotherapy.

Abstract IA16: Immune suppression in cancer and strategies for its reversal

Abstract The microenvironment is involved in multiple aspect of cancer progression. Tumor metastasis is a critical step in the progression of solid tumors that is associated with patient mortality, and the metastatic microenvironment is key regulator of this process. The pre-metastatic niche is the microenvironment important for metastatic initiation that is established at distant sites in response to primary tumor factors during cancer progression. We characterized this microenvironment which involves changes in both stromal and immune populations in the lungs of sarcoma-bearing mice and in the liver in pancreatic- bearing mice by flow cytometry and RNA sequencing approaches. We identified a gene signature in pre-metastatic niche formation that demonstrates upregulation of immune suppression genes that is consistent across different metastatic tissue including lung and liver as well as across species with commonalities in murine and human early metastatic microenvironments. Performing single cell RNA sequencing of the pre-metastatic niche revealed key immune suppressive genes were found in the myeloid cell clusters. In addition to the increase of myeloid cells and immunosuppressive pathways, we discovered that T cell populations are reduced in pre-metastatic lungs. We hypothesized that reversing this immunosuppressive environment would restore T cell function and antitumor immunity. We designed a novel approach in which we generated Genetically-Engineered Myeloid cells (GEMys) to deliver IL-12, a potent antitumor molecule, into the pre-metastatic microenvironment. We evaluated the lungs by flow cytometry and observed that IL12-GEMy-treated mice had increased numbers of T cells and enhanced expression of activation markers, resulting in reduced metastasis and increased survival. This model was effective in an aggressive experimental metastasis model of pancreatic liver metastasis and was not only able to limit metastatic progression but was able to cure a subset of mice compared to rapid metastatic progression in the liver within a month in the untreated pancreatic cancer mice. When combined with chemotherapy pre-conditioning, IL12-GEMys cured mice of established tumors and generated long-lived T cell memory, as these mice were immune to subsequent tumor challenge. These studies demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer. Citation Format: Rosandra N. Kaplan. Immune suppression in cancer and strategies for its reversal [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr IA16.

GDPLichi: a DNA Damage Repair-Related Gene Classifier for Predicting Lung Adenocarcinoma Immune Checkpoint Inhibitors Response.

Lung cancer is one of the most common and mortal malignancies, usually with a poor prognosis in its advanced or recurrent stages. Recently, immune checkpoint inhibitors (ICIs) immunotherapy has revolutionized the treatment of human cancers including lung adenocarcinoma (LUAD), and significantly improved patients’ prognoses. However, the prognostic and predictive outcomes differ because of tumor heterogeneity. Here, we present an effective method, GDPLichi (Genes of DNA damage repair to predict LUAD immune checkpoint inhibitors response), as the signature to predict the LUAD patient’s response to the ICIs. GDPLichi utilized only 7 maker genes from 8 DDR pathways to construct the predictive model and classified LUAD patients into two subgroups: low- and high-risk groups. The high-risk group was featured by worse prognosis and decreased B cells, CD8+ T cells, CD8+ central memory T cells, hematopoietic stem cells (HSC), myeloid dendritic cells (MDC), and immune scores as compared to the low-risk group. However, our research also suggests that the high-risk group was more sensitive to ICIs, which might be explained by increased TMB, neoantigen, immune checkpoint molecules, and immune suppression genes’ expression, but lower TIDE score as compared to the low-risk group. This conclusion was verified in three other LUAD cohort datasets (GSE30219, GSE31210, GSE50081).

Variants: Preferential Expression of Immune Suppressive Genes Contributes to SARS-CoV-2 increased Transmission and Virulence

Variants: Preferential Expression of Immune Suppressive Genes Contributes to SARS-CoV-2 increased Transmission and Virulence