NK Cell Infiltration Research Articles

Knocking down of Xkr8 enhances chemotherapy efficacy through modulating tumor immune microenvironment

Scramblase Xk-related protein 8 (Xkr8) regulates the externalization of phosphatidylserine (PS) during apoptosis and holds a pivotal role in fostering tumor immunosuppression. Targeting Xkr8 in conjunction with chemotherapy demonstrated a novel avenue for amplifying antitumor immune response and overcoming chemo-immune resistance. Here we further evaluated this strategy by using a clinically relevant orthotopic model and elucidated the mechanism through in-depth single-cell RNA sequencing (scRNA-seq). We found that Xkr8 knockdown exhibited the potential to lead to immunogenic cell death (ICD) by impeding the normal clearance of apoptotic cells. Co-delivery of Xkr8 small interference RNA (siRNA) and a prodrug conjugate of 5-fluorouracil (5-Fu) and oxoplatin (FuOXP) showed remarkable therapeutic efficacy in an orthotopic pancreatic tumor model with increased infiltration of proliferative NK cells and activated macrophages in the tumor microenvironment (TME). Single-cell trajectory analysis further unveiled that tumor infiltrating CD8+ T cells are differentiated favorably to cytotoxic over exhausted phenotype after combination treatment. Our study sheds new light on the impact of Xkr8 knockdown on TME and solidifies the rationale of combining Xkr8 knockdown with chemotherapy to treat various types of cancers.

PVALB Was Identified as an Independent Prognostic Factor for HCC Closely Related to Immunity, and Its Absence Accelerates Tumor Progression by Regulating NK Cell Infiltration.

Hepatocellular carcinoma is the most common primary liver cancer, with poor prognosis. Complex immune microenvironment of the liver is linked to the development of HCC. PVALB is a calcium-binding protein which has been described as a cancer suppressor gene in thyroid cancer and glioma. Nevertheless, the role of PVALB in HCC is unknown. We obtained data from TCGA and GSE54236 datasets. MCP-counter, WGCNA and LASSO model were applied to identify PVALB. With UALCAN, MethSurv, and other websites, we probed the expression, methylation and survival of PVALB. LinkedOmics and GSEA were adopted for functional analysis, while TIMER, TISIDB, Kaplan-Meier plotter, TIDE databases were utilized to evaluate the relevance of PVALB to the tumor immune microenvironment and predict immunotherapy efficacy. TargetScan, DIANA, LncRNASNP2 databases and relevant experiments were employed to construct ceRNA network. Finally, molecular docking and drug sensitivity of PVALB were characterized by GeneMANIA, CTD, and so on. PVALB was recognized as a gene associated with HCC and NK cell. Its expression was down-regulated in HCC tissue, which lead to adverse prognosis. Besides, the hypomethylation of PVALB was related to its reduced expression. Notably, PVALB was tightly linked to immune, and its reduced expression attenuated the anticancer effect of NK cells via the Fas/FasL pathway, leading to a adverse outcome. The lnc-YY1AP1-3/hsa-miR-6735-5p/PVALB axis may regulate the PVALB expression. Finally, we found immunotherapy might be a viable treatment option. In a word, PVALB is a prognostic indicator, whose low expression facilitates HCC progression by impacting NK cell infiltration.

The Immune Microenvironment Landscape of Pituitary NeuroEndocrine Tumors, a Transcriptomic Approach.

Pituitary neuroendocrine tumors (PitNET) are known to be variably infiltrated by different immune cells. Nonetheless, their role in pituitary oncogenesis has only begun to be unveiled. The immune microenvironment could determine the biological and clinical behavior of a neoplasm and may have prognostic implications. To evaluate the expression of immune-related genes and to correlate such expression with the presence of infiltrating immune cells in forty-two PitNETs of different lineages, we performed whole transcriptome analysis and RT-qPCR. Deconvolution analysis was carried out to infer the immune cell types present in each tumor and the presence of immune cells was confirmed by immunofluorescence. We found characteristic expression profiles of immune-related genes including those encoding interleukins and chemokines for each tumor lineage. Genes such as IL4-I1, IL-36A, TIRAP, IL-17REL, and CCL5 were upregulated in all PitNETS, whereas IL34, IL20RA, and IL-2RB characterize the NR5A1-, TBX19-, and POU1F1-derived tumors, respectively. Transcriptome deconvolution analysis showed that M2 macrophages, CD4+ T cells, CD8+ T cells, NK cells, and neutrophils can potentially infiltrate PitNET. Furthermore, CD4+ and CD8+ T cells and NK cells infiltration was validated by immunofluorescence. Expression of CCL18, IL-5RA, and HLA-B as well as macrophage tumor infiltration could identify patients who can potentially benefit from treatment with immune checkpoint inhibitors.

Multi-Omics Pan-Cancer Analysis of Procollagen N-Propeptidase Gene Family of ADAMTS as Novel Biomarkers to Associate with Prognosis, Tumor Immune Microenvironment, Signaling Pathways, and Drug Sensitivities.

The extracellular matrix (ECM) modeling induced by the metalloproteinases is a vital characteristic for tumor progression. Previous studies mainly focus on the functions of two subgroups of metalloproteinases: matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) in tumors. The roles of another important group: the ADAMs with thrombospondin motifs (ADAMTS) remain unclear. This study aimed to perform a pan-cancer analysis of procollagen N-propeptidase subgroup of ADAMTS (PNPSA). We systematically analyzed expression landscape, genomic variations, prognostic value, and cell expression clusters of PNPSA in pan-cancer based on the multiple integrated open databases. Besides, we also analyzed the impacts of expressions and genomic variations of PNPSA members on tumor immune microenvironment (TIME) and immune-related molecules in pan-cancer based on the immune-related open databases. The Gene Set Variation Analysis (GSVA) was performed to evaluate the associations of the whole PNPSA with prognosis, tumor indicators, TIME, and drug sensitivities. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to reveal related signaling pathways. Finally, immunohistochemical staining was used to validate the differential analysis results. We found a dual prognostic role of PNPSA members in pan-cancer and they were significantly correlated with TIME and immune-related molecules. Interestingly, the copy number variations (CNVs) of all PNPSA members were revealed to be negatively correlated with NK cell infiltration in most cancers. Single-cell sequencing analysis reveals expressions of PNPSA gene family members on some specific tumor and immune cells in addition to the fibroblasts. The GSVA score was found to have some predictive value for survival status in Brain Lower Grade Glioma (LGG), Mesothelioma (MESO), and Uveal Melanoma (UVM) and to be significantly correlated with tumorigenesis-related pathways such as PI3K-Akt, AGE-RAGE, etc. The GSVA score also shows some predictive value for chemotherapy and immunotherapy efficacy in some tumors. PNPSA was correlated with tumor development and might be potential tumor biomarker and therapeutic target.

Ischemia-free liver transplantation improves the prognosis of recipients using functionally marginal liver grafts.

The shortage of donor liver hinders the development of liver transplantation. This study aimed to clarify the poor outcomes of functionally marginal liver grafts (FMLs) and provide evidence for the improvement of ischemia-free liver transplantation (IFLT) after FML transplantation. Propensity score matching was used to control for confounding factors. The outcomes of the control group and FML group were compared to demonstrate the negative impact of FMLs on liver transplantation patients. We compared the clinical improvements of the different surgical types. To elucidate the underlying mechanism, we conducted bioinformatic analysis based on transcriptome and single-cell profiles. FMLs had a significantly greater hazard ratio (HR: 1.969, P=0.018) than did other marginal livers. A worse 90-day survival (Mortality: 12.3% vs. 5.0%, P=0.007) was observed in patients who underwent FML transplantation. Patients who received FMLs had a significant improvement in overall survival after IFLT (Mortality: 10.4% vs 31.3%, P=0.006). Pyroptosis and inflammation were inhibited in patients who underwent IFLT. The infiltration of natural killer cells was lower in liver grafts from these patients. Bulk transcriptome profiles revealed a positive relationship between IL-32 and Caspase 1 (R=0.73, P=0.01) and between IL-32 and Gasdermin D (R=0.84, P=0.0012). FML is a more important negative prognostic parameter than other marginal liver parameters. IFLT might ameliorate liver injury in FMLs by inhibiting the infiltration of NK cells, consequently leading to the abortion of IL-32, which drives pyroptosis in monocytes and macrophages.

BCL9 is a Risk Factor of Neck Lymph Nodes Metastasis and Correlated with Immune Cell Infiltration in Papillary Thyroid Carcinoma.

B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, contributed to tumor progression and metastasis in various tumors, whereas, the role of BCL9 in papillary thyroid cancer (PTC) has not been investigated. We acquired PTC gene expression data from The Cancer Genome Atlas (TCGA) database. Fifty-nine PTC tissues were applied to validate the clinical significance of BCL9. Cell experiments were applied to investigate the role of BCL9. Bioinformatics analysis was employed to investigate the biological functions of BCL9. We found that BCL9 was higher expressed (P < 0.05) and an independent risk factor for lymph node metastasis (OR = 3.770, P = 0.025), as well as associated with poorer progression-free survival (PFS) (P = 0.049) in PTC. BCL9 knockdown inhibited proliferation and invasion of PTC cells. BCL9 was positively associated with the key genes of Wnt/β-catenin and MAPK pathway by co-expression analysis. GO, KEGG and GSEA analysis showed BCL9 might participated in PPAR, cAMP, and focal adhesion pathway. CIBERSORT analysis found BCL9 was negatively associated with CD8+ T cells and NK cell infiltration and positively with PD-L1 expression. Therefore, BCL9 was associated with lymph node metastasis and shorter PFS of PTC, due to promotion of PTC cell proliferation and invasion, activation of Wnt/β-catenin and MAPK pathway, inhibition of CD8+ T and NK cell infiltration, and promotion of PD-L1 expression.

Abstract 5519: Enlarged metastasis-free tumor-draining lymph node is a marker of tumor invasion in local tissues with an active NK cell-mediated immunity

Abstract Background: Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for about 10% of all new cancer cases. The tumor-draining lymph node (TDLN) is a key site for the development of anti-tumor immunity that has been reported to be involved in both CD8+ T and NK cells. As a major role of innate immunity, NK cells can produce IFN-γ, perforin and granzyme to eliminate tumor cells in the early stages of cancer development. This study aimed to investigate the difference of clinicopathological and immunological profiles in local tissues between CRC patients with enlarged and non-enlarged metastasis-free TDLNs. Methods: The patient cohort was obtained from the NEPDC cohort (No. 2015CB554000). A total of 45 patients with pathologically confirmed stage I-II colorectal cancer were included in this study. Based on the MRI, these patients were divided into enlarged and non-enlarged TDLN groups (lymph nodes &amp;gt;/= 5mm and lymph nodes &amp;lt; 5mm). We collected the tumor tissue and tumor-adjacent normal tissue that was 2 cm away from the tumor lesion. The genomic DNA of the tumor and tumor-adjacent normal tissues was treated with bisulfite and analyzed with the EPIC methylation array. The MeTMEC algorithm based on the methylation profile estimated subsequent cell abundance in these patient tissues. Results: The study sample was comprised of 22 enlarged TDLN and 23 non-enlarged TDLN patients. There were 4 T1-T2 and 18 T3-T4 patients in the enlarged TDLN group, with 12 T1-T2 and 11 T3-T4 patients in the non-enlarged TDLN group. The proportion of T3-T4 patients in the enlarged TDLN group was higher than that in the non-enlarged TDLN group (p=0.017, Chi-squared test), suggesting an advanced invasion in the local tissue of patients with enlarged TDLN. Then, the abundance of monocytes, dendritic cells, macrophages, neutrophils, eosinophils, Treg cells, naive T cells, memory T cells, CD8+ T cells, NK cells, and B cells in the tumor and tumor-adjacent normal tissues were inferred with the MeTMEC algorithm by using DNA methylation profile. In the T1-T2 subset, we found that the infiltration of NK cells in the tumor-adjacent normal tissues was significantly more abundant in the enlarged TDLN group compared with the non-enlarged TDLN group (p=0.021, t-test), suggesting an active immune response of NK cells for potential local tumor control. No significant difference in other immune cells was observed between the two groups. Conclusion: Enlarged TDLN indicates an advanced invasion of tumors in local tissues with an active NK cell-mediated immunity. Our findings suggest that enlarged metastasis-free TDLN might be a promising marker for NK cell-based cancer immunotherapy. Key words: Colorectal Cancer, Lymph Nodes, Natural Killer Cells Citation Format: Zixiao Wan, Huichuan Yu. Enlarged metastasis-free tumor-draining lymph node is a marker of tumor invasion in local tissues with an active NK cell-mediated immunity [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 5519.

Abstract 3847: Dual targeting of EGFL6 and VEGF-A to overcome immune suppression in ovarian cancer

Abstract Background: Ovarian carcinomas are known to have a high degree of immune suppression, which may be related, in part, to high levels of vascular endothelial growth factor-A (VEGF-A). Bevacizumab (anti-VEGF antibody) has been shown to increase infiltration of T-cells in the tumor microenvironment (TME). We have also identified EGFL6 as an important angiogenic protein. Here, we examined the biological effects of blocking EGFL6 and VEGF-A in ovarian cancer models. Methods: We studied the in vitro (endothelial tube formation and migration assays) and in vivo (ID8KO-Trp53;Brca2 and SKOV3-ip1 models) biological effects of EGFL6 and VEGF-A blockade. Tumor-infiltrating immune cells were studied by flow cytometry and immunohistochemistry staining. Results: Blocking EGFL6 and VEGF-A simultaneously with monoclonal antibodies in RF24 endothelial cells decreased tube formation and migration significantly, in comparison to VEGF-A blockade alone (p&amp;lt;0.001). EGFL6 monotherapy in ID8KO-Trp53;Brca2 tumor bearing syngeneic mice decreased tumor burden (~2.5 fold, p&amp;lt;0.0001) and increased the infiltration of IFNγ+ (~2.75 fold, p&amp;lt;0.0001) and GzmB+ (~1.75 fold, p&amp;lt;0.01) CD8+ T-cells and IFNγ+ (~2 fold, p&amp;lt;0.0001) and GzmB+ (~2 fold, p&amp;lt;0.01) CD4+ T-cells while decreasing PD-L1+ cells (~1.8 fold, p=0.01). Dual blockade of EGFL6 and VEGF-A in SKOV3-ip1 tumor bearing nude mice significantly decreased tumor burden (~3.72 fold, p&amp;lt;0.01) and angiogenesis (~2.36 fold, p&amp;lt;0.05) compared to controls and single-agent blockade. Similar blockade in ID8KO-Trp53;Brca2 tumor bearing mice significantly decreased tumor burden (~4.2 fold, p=0.0004), improved CD8+ (~3.32 fold, p=0.0004) and CD4+ (~2.81 fold, p&amp;lt;0.0001) T-cell infiltration, NK cell infiltration (~2.3 fold, p=0.0005) and decreased myeloid-derived suppressor cells (MDSCs) (~1.92 fold, p=0.0278) compared to EGFL6 and VEGF-A blockade alone. Conclusions: Our results demonstrate that simultaneously targeting the angiogenesis drivers EGFL6 and VEGF-A in ovarian cancer significantly decreased tumor burden in vivo and increased the infiltration of T lymphocytes compared to single-agent blockade. AACR Category: Tumor Microenvironment and Immune Oncology Key words: ovarian cancer, immune suppression, angiogenesis, VEGF-A, EGFL6 Citation Format: Sujanitha Umamaheswaran, Lingegowda S. Mangala, Robiya Joseph, Nicholas B. Jennings, Mark S. Kim, Yunfei Wen, Emine Bayraktar, Ningyan Zhang, Zhiqiang An, Anil K. Sood. Dual targeting of EGFL6 and VEGF-A to overcome immune suppression in ovarian 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 3847.

Abstract 2687: NK cell plasticity in SCLC is linked to tumor transcriptional diversity

Abstract Natural Killer (NK) cells are innate lymphoid cells that are pivotal in the body’s response to cancer. To harness their cytotoxicity in clinical interventions, it is critical to understand their biology, which is highly tissue- and tumor-specific. In Small Cell Lung Cancer (SCLC), reports have described widely divergent levels of NK activity in neuroendocrine (NE) and non-neuroendocrine (non-NE) subtypes. Diminished self-antigen presentation by MHC-I is a classic trigger of NK cell activity but, counter-intuitively, NK cells exhibit reduced cytotoxicity in MHC-I downregulated NE SCLC. To explore potential causes for depressed NK activity in NE SCLC relative to non-NE, we applied scRNA-Seq to 45 SCLC patient biopsies and rapid autopsies, and identified 4000 NK cells based on gene expression signature. There was a notable dip in NK cell infiltration in NE tumors compared to non-NE tumors. Tumor cell-surface ligands recognized by activating and, unexpectedly, inhibitory NK cell receptors were generally both more highly expressed in non-NE tumors. While inhibitory NK receptors were comparable in NE and non-NE tumors, activating NK receptors were expressed significantly, though modestly, higher in non-NE. We observed tumor site-specific gene expression patterns in NK cells from different metastatic sites. Overall, there were a remarkably high number of differentially expressed genes (&amp;gt;1k) between NK cells from NE and non-NE tumors, even after controlling for tumor site. Together, our work suggests that NK cell plasticity is manifest at the level of SCLC transcriptional subtypes. Citation Format: Justin Malin, Sophie Zhuang, Ajit Kumar Sharma, Joseph Clara, Anish Thomas. NK cell plasticity in SCLC is linked to tumor transcriptional diversity [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 2687.

Abstract 5537: How aging promotes immune mediated reactivation from metastatic melanoma dormancy

Abstract Disseminated cancer cells that escape the primary tumor can seed distal tissues but may take several years to grow into clinically detectable metastases, a phenomenon termed tumor dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully model melanoma dormancy. We have previously shown that while tumors grow more slowly in aged mouse skin, they form more lung metastases in aged mice compared to young mice. We queried whether the lung microenvironment undergoes age-related changes that establish a permissive niche for metastatic outgrowth. We identified that Wnt5A and AXL are not only required for efficient dissemination away from the primary tumor but also activate dormancy of melanoma cells within the lung. The young lung microenvironment maintains melanoma cells in a dormant state; however, age-associated reprograming of lung fibroblasts increases secretion of the soluble Wnt antagonist sFRP1, which inhibits both Wnt5A and AXL to enable reactivation from dormancy and the formation of larger metastatic colonies in the aged lung. We found that downregulation of AXL in melanoma cells leads to an increase in the related TAM family receptor MERTK, which promotes a proliferative melanoma phenotype and enables reactivation from dormancy. We have now found that both the aged-pre and post metastatic niche regulate immune cells in the lung to create a permissive niche for reactivation of metastatic melanoma. NK cells are significantly decreased in the healthy lungs of aged mice, are significantly less active, and have increased exhaustion markers. NK cell infiltration remains low in early and late-stage metastasis in aged mice. Depletion of NK cells in young mice is sufficient to induce reactivation from dormancy. Further, MERTK induced reactivation of melanoma cells results in paracrine secretion of its own ligand PROS1 to facilitate a growth permissive lung microenvironment. Melanoma-secreted PROS1 increases infiltration of PMN-MDSCs and decreases macrophage infiltration to maintain an immunosuppressive lung niche for efficient outgrowth. Selective inhibition of PMN-MDSCs using a TRIAL-R2 agonistic antibody significantly decreases metastatic outgrowth and promotes smaller, non-proliferative dormant colonies. Our findings indicate a significant role that aging plays in altering NK-cell infiltration and function to create a growth permissive pre-metastatic niche and how reactivation from dormancy can further contribute to create an immunosuppressive microenvironment for efficient outgrowth via PROS1 induced infiltration of PMN-MDSCs. Importantly, the clear differences found in our young and aged mouse modelling systems for outgrowth in metastatic tissues necessitates the need for age-appropriate pre-clinical modelling for better patient and disease representation. Citation Format: Pulkit Datt, Jhon Passamonte, Christopher Price, Daniel Zabransky, Yash Chhabra, Ashani Weeraratna, Mitchell E. Fane. How aging promotes immune mediated reactivation from metastatic melanoma dormancy [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 5537.

Abstract 6875: PIM kinases alter the prostate tumor immune microenvironment

Abstract Prostate cancer is the second leading cause of cancer-related deaths in American men. While prostate cancer patients typically respond to androgen-deprivation therapy, androgen receptor signaling inhibitor (ARSI), and/or taxane chemotherapy, patients inevitably develop resistance, and the disease progresses to an untreatable and lethal form known as castrate resistant prostate cancer (CRPC). Immunotherapy has changed the treatment paradigm for many types of cancer; however, immune checkpoint inhibitors have not shown clinical benefits in CRPC. Clinical and preclinical evidence indicates that this lack of efficacy can largely be attributed to the immunosuppressive tumor microenvironment. Tumor associated macrophages (TAMs) secrete factors that promote tumor progression and suppress antitumor immunity, making them a promising therapeutic target. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases are serine/threonine kinases that are overexpressed in prostate cancer. PIM regulates many signaling pathways that promote cell proliferation and survival. However, how PIM kinase alters the prostate tumor immune microenvironment and impacts immunotherapy resistance is not well understood. We analyzed primary prostate and metastatic lymph node samples from treatment-naive metastatic hormone-sensitive prostate cancer patients. Prostate cancer samples with PIM1/PIM2/PIM3-high and -low expression were classified by top and bottom quartile, respectively. PIM high tumors had higher expression of immunostimulatory genes (IL1β, TNF, and TNFSF13), increased infiltration of M2 macrophages, B cells, and NK Cells, and a higher T cell inflamed score compared to PIM-low tumors. Our results demonstrate that PIM overexpressing prostate tumors display increased inflammation and infiltration of immunosuppressive immune cells, including M2 macrophages. Due to the changes observed in inflammation, we hypothesized that PIM kinase may regulate macrophage inflammatory signaling. We treated bone marrow derived macrophages with a PIM kinase inhibitor and showed that PIM inhibition suppresses inflammasome signaling and the release of the pro-inflammatory cytokine, IL-1β. Chronic inflammation can lead to the recruitment of TAMs, further promoting resistance to immune checkpoint inhibitors. Utilizing a syngeneic mouse model of prostate cancer, we demonstrated that PIM inhibition in combination with immune checkpoint blockade synergistically decreased tumor growth. Furthermore, immunoprofiling demonstrated that combination treatment enhances T cell activity. Overall, our results suggest that PIM kinase plays an important role in regulating the prostate tumor immune microenvironment and that PIM kinase may be a potential target to enhance the efficacy of immunotherapy for the treatment of prostate cancer. Citation Format: Amber N. Clements, Kai Sutterby, Sachin Kumar Deshmukh, Sharon Wu, Joanne Xiu, Alex Farrell, Milan Radovich, Chadi Nabhan, Elisabeth I. Heath, Rana R. McKay, Alejandro Recio-Boiles, Noel A. Warfel. PIM kinases alter the prostate tumor immune microenvironment [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 6875.

Abstract 5201: Characterization of PDLIM2 in non-small cell lung cancer

Abstract Introduction: PDZ and LIM domain protein 2 (PDLIM2) acts as tumor suppressor by downregulating NF-κB and STAT3 signaling, modulating inflammation, immune response, and cell survival. Mouse model studies have demonstrated that downregulation of PDLIM2 leads to PD-1 immune blockade and chemotherapy resistance. We present a large clinical and molecular characterization of PDLIM2 expression in non-small cell lung cancer (NSCLC). Methods: NextGen sequencing of DNA (whole exome)/RNA (whole transcriptome) was performed for NSCLC (Total N = 29126; Adenocarcinoma [-A, N = 15765], Squamous [-S, N = 6416]) patient tumors submitted to Caris Life Sciences (Phoenix, AZ). Mutations were defined as pathogenic SNVs/indels. Samples were stratified by PDLIM2 expression quartiles (in transcripts per million [TPM]) for all NSCLC tumors (Q4: H, Q1: L). PD-L1 expression [22C3; Positive (+): tumor proportion score (TPS) ≥1%]was assessed by IHC. High tumor mutational burden (TMB-high) set as ≥10 mutations per Mb. Cell infiltration in the tumor microenvironment was estimated by QuantiSEQ. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (T cell-inflamed). Real-world overall survival was assessed from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined subpopulations. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons (p &amp;lt; 0.05). Results: Median PDLIM2 expression was higher in NSCLC-A compared to NSCLC-S (15.3 vs 13.6 TPM, p &amp;lt; 0.001). PDLIM2-AL had a lower prevalence of EGFR mutations than PDLIM2-AH (15.5% vs 19.4%, p &amp;lt; 0.01), but the opposite pattern was observed for STK11 (21.5% vs 11.2%), RB1 (7.5% vs 3.8%), TP53 (62.9% vs 52.8%), KEAP1 (18.4% vs 10.9%), and SMARCA4 (9.15 vs 4.5%) alterations (all p &amp;lt; 0.001). PDLIM2-AL tumors had decreased PD-L1+ (L: 52.4% vs H: 58.4% p &amp;lt; 0.001) but increased TMB-High status (L: 40.9% vs H: 28.5%, p &amp;lt; 0.001). PD-L1+ and TMB-H frequency did not vary with PDLIM2 expression in NSCLC-S. In both NSCLC-A and -S tumors, PDLIM2H had increased infiltration of NK cells, macrophages, dendritic cells, T cells, neutrophils, monocytes, and B cells compared to PDLIM2L (all p &amp;lt; 0.05), in addition to increased T cell-inflammed scores (p &amp;lt; 0.001). PDLIM2-AH was associated with improved overall survival (OS) (median 21.7 vs L: 15.6 months; p &amp;lt; 0.001; Hazard ratio [HR] = 0.825, 95% Confidence Interval [CI] 0.765 - 0.889) and time on pembrolizumab treatment (median 6.2 vs L: 5.6 months; p = 0.009; HR = 0.825 95% CI 0.714 - 0.953). No difference in OS was observed between PDLIM2-SH vs L tumors (H: 14.8 vs L: 14.0 months; p = 0.37; HR = 0.95, CI 0.85-1.06). Conclusions: NSCLC-A with high PDLIM2 expression have a unique mutational profile, increased immune cell infiltration and favorable OS. Therapeutic strategies for targeting PDLIM2 to modulate NF-κB and STAT3 signaling should be further explored. Citation Format: Karam Ashouri, Harris Krause, Andrew Elliott, Stephen V. Liu, Patrick C. Ma, Balazs Halmos, Zhaoxia Qu, Gutian Xiao, Ari Vanderwalde, Jorge J. Nieva. Characterization of PDLIM2 in non-small cell lung 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 5201.

Abstract 5272: APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells

Abstract Introduction: Treatment approach combining cancer cell targeted therapy and immunotherapy is an effective strategy to trigger robust tumor-specific immune responses. The aim of this study is to determine the impact of APL-1202, a clinical-stage drug candidate (NCT04813107), on cellular stress-driven immunogenic outcomes in bladder cancer cells. Methods: RNA-seq analysis was performed using bladder cancer and endothelial cell lines (A56, J82, T24, HUVEC) treated with APL-1202. Key determinants of immunogenic cell death pathway were measured by cell-surface translocation of calreticulin (flow cytometry), extracellular ATP release assay (bioluminescence) and HMGB1 secretion (ELISA) in the supernatants of cancer cells after APL-1202 treatment. Effects on the innate immune effector function was measured by flow cytometry-based phagocytosis assay consisting of Far-Red dye-stained, pre-treated bladder cancer cell lines (UMUC3, T24) in coculture with THP1-derived macrophages/immature dendritic cells or human monocyte-derived dendritic cells. In vivo, tumor end-point immune cell analysis, tumor weight and mouse survival were compared in chemically induced (BBN: N-butyl-N-(4-hydroxybutyl)-nitrosamine) orthotopic bladder cancer mice treated with APL-1202 and anti-PD1 antibody. Results: Pathway analysis of RNA-seq data from APL-1202 treated cells suggested upregulation of cellular stress-related gene signatures (oxidative stress, autophagy, P53) and pro-inflammatory responses (IL-6, IL-8, HMGB1 signaling). Conversely, downregulation of cell cycle (E2F targets, G2-M checkpoint) and IL-10 signaling suggested increased genomic stress and inflammatory switching in the treated cancer cells. Acute exposure to APL-1202 increased cell-surface translocation of calreticulin as well as extracellular release of ATP and HMGB1 in the treated T24 and UMUC3 cells. Higher percentages of phagocytic activities were detected in both THP-1-derived macrophages/immature dendritic cells and human monocyte-derived dendritic cells after coculture with APL-1202 treated T24 and UMUC3 cells compared to coculture with DMSO treated control cells. Immune phenotyping in tumors derived from BBN bladder cancer mouse model revealed increased infiltration of NK cells and CD8+ T cells. Furthermore, APL-1202 enhanced the anti-tumor effects of a therapeutic anti-PD1 antibody in mice as reflected by significantly decreased tumor weights and increased survival in a combination group with APL-1202 treatment compared to the anti-PD-1 antibody alone treatment group. Conclusion: APL-1202 may improve the efficacy of immune-checkpoint blockade therapies in bladder cancer patients by enhancing cancer cell immunogenicity and subsequent anti-tumor effector immune cell processes, particularly in tumors with deficits in antigen-specific immunity. Citation Format: Rakesh BAM, Jingmin Guan, Neetha Nanoth Vellichirammal, Murli Manohar, David Mulholland, John Sfakianos, Qiaoling Sun, Zuoan Yi, Alice Chen. APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells [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 5272.

Abstract 162: Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer

Abstract Background: Immune checkpoint blockade (ICB), which unleashes potent CD8+ T cell-based anti-tumor responses, has emerged as a promising therapeutic option for several cancer types. However, response rates stay limited in gastric cancer (GC) patients, indicating the necessity of combinational strategies to overcome other immunosuppressive factors. Fatty acid synthesis (FAS) is an indispensable metabolic pathway proven to support cancer cell survival. The rate-limiting enzyme of FAS, FASN, is upregulated in several cancers and could contribute to cancer immune escape. Though some investigations propose that specific gene alterations can cause FASN upregulation under the GC setting, the immunoregulatory role and therapeutic value of FASN in the tumor microenvironment (TME) of GC have not been fully elucidated. Methods: FASN expression was measured by Western Blotting and flow cytometry. Bioinformatic analysis was conducted via an online website (www.camoip.net). The syngeneic GC mouse model was established by subcutaneous inoculation of MFC cells in 615-line mice, treated by vehicles, FASN inhibitor (FASNi), anti-PD-1 antibodies, or FASNi+anti-PD-1 antibodies. TAM and CD8+ T cell depletion was implemented by anti-CSF1R and anti-CD8α antibodies. Bone marrow-derived macrophages (BMDMs) were extracted from the femurs of 615-line mice, induced by M-CSF and stimulated by LPS or IL-4. The proportions and functional status of immune cells were identified by flow cytometry. Results: FASN was upregulated in GC tissues and GC human cell lines. Bioinformatic analyses demonstrated that FASN expression was related to poor infiltration of cytotoxic lymphocytes, as well as the downregulation of pathways related to anti-cancer immune responses. FASNi inhibited tumor growth in a syngeneic GC mouse model, with elevated PD-1 and IFN-γ expression in CD8+ T cells and increased NK cell infiltration, suggesting the enhancement in anti-cancer cytotoxicity. Meanwhile, FASNi induced a shift from M2-like to M1-like in tumor-associated macrophages (TAM). We detected that M2-like TAM and CD8+ T cells harbored higher FASN expression than other immune cells in GC mouse tissues, and tumor-suppressing effects of FASN inhibition could be reversed by TAM and CD8+ T cell depletion, which corroborated the key role of TAM and CD8+ T cells in shaping FASN-mediated tumor regulation. FASNi could also repolarize BMDMs from M1-like to M2-like in vitro. Finally, FASNi exhibited synergistic tumor control with anti-PD-1 therapy in GC and melanoma mouse models. Conclusion: This work sheds light on the impact of FASN on TME modulation of GC. We proved that FASNi could repolarize M2-like TAM to M1 phenotypes and promote cytotoxic lymphocyte-mediated anti-tumor immunity. Our results propose FASN inhibition as a potential therapeutic strategy to sensitize ICB therapy in GC. Citation Format: Hanbing Wang, Tao Shi, Yue Wang, Yuting Luo, Yunfeng Pan, Xiaoyu Zhou, Yue Zhang, Baorui Liu, Jia Wei. Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric 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 162.

Prevention and treatment of HPV-related cancer through a mRNA vaccine expressing APC-targeting antigen.

Persistent human papillomavirus (HPV) infection is associated with multiple malignancies. Developing therapeutic vaccines to eliminate HPV-infected and malignant cells holds significant value. In this study, we introduced a lipid nanoparticle encapsulated mRNA vaccine expressing tHA-mE7-mE6. Mutations were introduced into E6 and E7 of HPV to eliminate their tumourigenicity. A truncated influenza haemagglutinin protein (tHA), which binds to the CD209 receptor on the surface of dendritic cells (DCs), was fused with mE7-mE6 in order to allow efficient uptake of antigen by antigen presenting cells. The tHA-mE7-mE6 (mRNA) showed higher therapeutic efficacy than mE7-mE6 (mRNA) in an E6 and E7+ tumour model. The treatment resulted in complete tumour regression and prevented tumour formation. Strong CD8+ T-cell immune response was induced, contributing to preventing and curing of E6 and E7+ tumour. Antigen-specific CD8+ T were found in spleens, peripheral blood and in tumours. In addition, the tumour infiltration of DC and NK cells were increased post therapy. In conclusion, this study described a therapeutic mRNA vaccine inducing strong anti-tumour immunity in peripheral and in tumour microenvironment, holding promising potential to treat HPV-induced cancer and to prevent cancer recurrence.

Immunohistochemical Evaluation of NKP46 Receptor Expression and the Number of NK Cells in the Endometrium of Patients with Endometriosis.

Endometriosis is a medical condition that can cause infertility in women. Women with endometriosis experience a decrease in NK cell cytotoxic activity against endometrial cells, ultimately contributing to the spread of these cells. To assess the frequency of NK cells and the expression of the NKP46 receptor in endometrial tissue from patients with endometriosis using immunohistochemistry. 30 endometrial tissue specimens were collected from three groups of cases with mild (n=11), moderate (n=10), and severe endometriosis (n=9), respectively. Additionally, 20 normal endometrial tissue specimens were collected as the control group. Immunohistochemical staining was carried out using specific human monoclonal antibodies against CD56 and NKP46 molecules. Cases with severe endometriosis had a significantly higher number of CD56+ uterine NK cells (26.19±2.50) compared to fertile women (15.02±0.622) and women with mild to moderate endometriosis (p<0.001). However, there was no significant difference between the mild to moderate patients compared with the healthy women (p>0.05). Endometrial NKp46 expression was lower in women with severe endometriosis (0.447±0.0829) compared to fertile women (0.987±0.115, p=0.03). The NKp46+/CD56+ cell ratio was also lower in women with severe endometriosis (0.019±0.003) compared to fertile women (0.072±0.011, p=0.01). Women with severe endometriosis demonstrated an increased rate of infiltrated uterine NK cells and a significant decrease in NKP46 expression compared to fertile women. Therefore, NK cells and the NKp46 receptor may be involved in the development of endometriosis.

Abstract B024: Immune exhaustion and reversal of progestin-related immune modulation in adaptive resistance to levonorgestrel intrauterine device for treatment of atypical hyperplasia and early endometrial cancer

Abstract Background: A growing proportion of women diagnosed with atypical hyperplasia (AH) and early-stage endometrioid endometrial cancer (EEC) require nonsurgical treatment options. Our previously published phase II clinical trial of the levonorgestrel intrauterine device (LIUD) for AH and grade 1 EEC (G1EEC) demonstrated 83% response rate at 1 year; however, prospective long-term follow-up data and determinants for duration of response to LIUD are limited. Methods: Follow-up data from patients that participated in our prospective phase II trial of LIUD as a nonsurgical approach for AH and G1EEC were collected from medical records. Duration of response and time to relapse were evaluated. Spatial transcriptomic profiling from longitudinal biopsy samples from pre-treatment (n=5), on-treatment (n=5), and disease relapse lesions (n=3) were evaluated using digital spatial profiling (DSP, Nanostring GeoMX Whole Transcriptome Atlas). In silico cell type deconvolution was performed and pathway analyses was conducted using gene set variation analysis enrichment. Results: Of 43 participants exhibiting initial response to LIUD, 41 had follow-up data. Sixteen experienced disease relapse (39%) with a median time to relapse of 17 months after initial response. Clinical factors associated with shorter duration of response included age (HR 0.95, p=0.021), initial diagnosis of G1EEC (HR 3.51, p=0.014), premenopausal status (HR 3.85, p=0.039), and Hispanic ethnicity (HR 3.45, p=0.017). Pre-treatment IGF1 and IGF2 expression levels were positive predictors of duration of response (HR 0.44 and 0.45, respectively, p&amp;lt;0.05). DSP results revealed that LIUD increased NK cells (log2FC=46.13, FDR=0.004), and lymphocyte cytotoxicity signaling markers GZMB (log2FC=1.06, padj=0.023) and PRF1 (log2FC=1.62, padj=0.004), but NK cells were reduced in relapse lesions (log2FC=-55.96, FDR=0.02) as was PRF1 expression (log2FC=-1.87, padj=0.038). Multiple immune-related pathways (IFNα and IFNγ response, TGFβ signaling) were significantly upregulated in relapse lesions compared to matched pre-treatment lesions (FDR&amp;lt;0.05). IDO1 expression, a marker for immune exhaustion, was upregulated in relapse lesions compared to matched pre-treatment and on-treatment lesions (log2FC=2.56, FDR=0.01). Conclusions: While LIUD for treatment of AH or G1EEC has excellent initial response rates, upfront resistance, and relapse after initial response to LIUD impacts nearly half of patients. Our study shows progestin-related immunomodulatory effects, including increased NK cell infiltration and lymphocyte cytotoxicity. Immune mechanisms for relapse are implicated here, including reversal of progestin-related immunomodulatory effects (decreased NK cell infiltration and lymphocyte cytotoxicity) and increased immune exhaustion (prolonged IFNα and IFNγ response and increased IDO1 expression in relapse lesions). Thus, relieving immune exhaustion may increase durability of response to LIUD for treatment of AH or G1EEC. Citation Format: Mikayla B. Bowen, Brenda Melendez, Qian Zhang, Richard Yang, Bryan Fellman, Barrett Lawson, Naomi Adjei, Joseph Celestino, Tri Nguyen, Khalida Wani, Bhavana Singh, Christof Straub, Liang Zhang, Cheryl Tan, Felicia New, Diana Urbauer, Alexander Lazar, Karen Lu, Jennifer Wargo, Shannon N. Westin, Melinda S. Yates. Immune exhaustion and reversal of progestin-related immune modulation in adaptive resistance to levonorgestrel intrauterine device for treatment of atypical hyperplasia and early endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B024.

Hybrid epithelial-mesenchymal status of lung cancer dictates metastatic success through differential interaction with NK cells

BackgroundEpithelial to mesenchymal transition (EMT) endows cancer cells with pro-metastatic properties, which appear most effective when cells enter an intermediate hybrid (H) state, characterized by integrated mesenchymal (M) and epithelial...

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Exploration of a hypoxia-immune-related microenvironment gene signature and prediction model for hepatitis C-induced early-stage fibrosis

BackgroundLiver fibrosis contributes to significant morbidity and mortality in Western nations, primarily attributed to chronic hepatitis C virus (HCV) infection. Hypoxia and immune status have been reported to be significantly correlated with the progression of liver fibrosis. The current research aimed to investigate the gene signature related to the hypoxia-immune-related microenvironment and identify potential targets for liver fibrosis.MethodSequencing data obtained from GEO were employed to assess the hypoxia and immune status of the discovery set utilizing UMAP and ESTIMATE methods. The prognostic genes were screened utilizing the LASSO model. The infiltration level of 22 types of immune cells was quantified utilizing CIBERSORT, and a prognosis-predictive model was established based on the selected genes. The model was also verified using qRT-PCR with surgical resection samples and liver failure samples RNA-sequencing data.ResultsElevated hypoxia and immune status were linked to an unfavorable prognosis in HCV-induced early-stage liver fibrosis. Increased plasma and resting NK cell infiltration were identified as a risk factor for liver fibrosis progression. Additionally, CYP1A2, CBS, GSTZ1, FOXA1, WDR72 and UHMK1 were determined as hypoxia-immune-related protective genes. The combined model effectively predicted patient prognosis. Furthermore, the preliminary validation of clinical samples supported most of the conclusions drawn from this study.ConclusionThe prognosis-predictive model developed using six hypoxia-immune-related genes effectively predicts the prognosis and progression of liver fibrosis. The current study opens new avenues for the future prediction and treatment of liver fibrosis.