Tumor-infiltrating Macrophages Research Articles

IL-33/NF-κB/ST2L/Rab37 positive-feedback loop promotes M2 macrophage to limit chemotherapeutic efficacy in lung cancer

IL-33 is a danger signal that binds to its receptor ST2L to promote tumor progression. This study identifies the IL-33/ST2L positive-feedback loop and the trafficking of ST2L membrane presentation in macrophages that contribute to lung tumor progression. Mechanistically, IL-33 induces ST2L upregulation by activating NF-κB, which binds to the promoter region of the ST2L gene. Moreover, Rab37, a small GTPase involved in membrane trafficking, mediates ST2L trafficking to the plasma membrane of M2 macrophages. This IL-33/NF-κB/ST2L/Rab37 axis promotes positive-feedback loops that enhance ST2L expression and membrane trafficking in M2 macrophages. Notably, neutralizing antibodies against IL-33 or ST2L block NF-κB activity, suppress M2 macrophage polarization, and synergistically inhibit tumor growth when combined with cisplatin treatment in vitro/vivo. Clinically, Rab37+/ST2L+/CD206+ tumor-infiltrating M2 macrophages correlate with advanced-stage lung cancer patients with poor response to chemotherapy. These findings unveil a positive-feedback mechanism and provide a basis for IL-33/ST2L-targeting therapy for cancer.

Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma.

We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Abstract PO2-18-05: Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model

Abstract Background Trastuzumab deruxtecan (T-DXd) administration improves response for patients with HER2-positive metastatic breast cancer (HER2+ BC). Unfortunately, 50% of patients relapse after 2 years. T-DXd resistance mechanisms are being explored. For trastuzumab we have shown that mucin 4 (MUC4) expression is an independent predictor of poor response in HER2+BC patients. MUC4 is upregulated by soluble TNF (sTNF) secreted by the tumor, confers resistance to trastuzumab by hiding its epitope on the HER2, reducing its binding and decreasing anti-tumor phagocytic function. In preclinical models of de novo trastuzumab-resistant tumors, combination of a sTNF blocking agent INB03, (DN), with T-DXd decreases tumor growth compared to T-DXd alone. To disclose the underlying mechanism, we studied whether DN improved internalization of T-DXd in tumor cells and modified the innate immune response to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Methods Nude mice bearing HER2+MUC4+ JIMT-1 tumor, primary resistant to trastuzumab, pertuzumab and lapatinib, were treated with IgG 5 mg/kg, T-DXd 5 mg/kg (T-DXd 5), 2.5 mg/kg (T-DXd 2.5) or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored. Mitotic figures/field (mean) were analyzed in H&E tumor sections. Tumor-infiltrating macrophages were studied by flow cytometry. IFNƔ was determined in tumor extracts by ELISA. Internalization of T-DXd in JIMT-1 cells was studied in a S1 Incucyte along 18h by Fab-rhodo red labeling. Results T-DXd dose-response curves showed inhibition in tumor growth of 83%, 61% and 37% for T-DXd 5, 2.5 and 1.25 mg/kg treatment respectively compared with IgG-treated tumors. DN alone exhibited no antitumor activity. T-DXd+DN increased the antitumor effect by 10%, 33% and 97% for T-DXd dose of 5, 2.5 and 1.25 mg/kg respectively. Tumor growth inhibition of T-DXd 5 was similar to combination of T-DXd1.25+DN. Addition of DN did not have toxicity. DN increases IFNƔ production in the TME of T-DXd treated tumors vs T-DXd alone and promoted macrophage recruitment to the tumor bed and polarization to the antitumor M1-like phenotype. Histopathological analysis of the tumor showed a significant decrease in proliferation in all the combined and in the T-DXd 5mg/kg (2.8-3.7 mitotic figures/field) vs IgG (6.0 mitotic figures/field). In vitro, JIMT-1 cells treated with DN internalized 40% more T-DXd than its vehicle-treated counterparts. Conclusions Neutralizing sTNF with DN enhances T-DXd effect in a multiple HER2 targeted therapy resistant model of MUC4 expressing HER2+ BC. Combination of DN with T-DXd increases tumor response at all dose levels tested. The largest effect was seen at lower doses (T-DXd 1.25+DN), which mimicked the effect of 4 times the dose of T-DXd alone, suggesting significant synergy with DN as dose of T-DXd decreases. Combination of DN with T-DXd in MUC4 expressing HER2+ BC improves response to T-DXd alone by increasing T-DXd internalization and improving anti-tumor innate immune responses in the TME without increasing toxicity. The results suggest this combination should be investigated in clinical trials in patients who have MUC4 expressing tumors when T-DXd is started or become resistant to T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Sofia Naveiro, Rosalia Cordo-Russo, Agustina Dupont, Mercogliano María Florencia, Roxana Schillaci. Blocking soluble TNF to Improve potency of trastuzumab deruxtecan by increasing internalization and antitumor innate immune response in a resistant HER2-positive breast cancer model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-05.

The effect of PHD inhibitor on tumor microenvironment and tumor immune response

Since the approval of HIF-PH inhibitors, HIF-PH inhibitors have been used clinically, and many studies and clinical case reports have been reported in Japan. A lot of information has been accumulated on clinical usage. However, HIF-PH inhibitors require careful administration for cancer patients due to their action mechanism through upregulating hypoxia-inducible factors (HIFs) level. In cancer cells, HIFs affect tumor progression and contribute to chemo- and radio-resistance. On the other hand, upregulation of HIFs in immune cells is associated with inflammation and suppress tumor progression. However, these controversial effects are not clear in in vivo model. It is needed to reveal whether upregulating HIFs level is beneficial for cancer therapy or not. We have previously reported that HIF-PH inhibitor treatment in tumor bearing mice model led to reconstitute tumor blood vessel and inhibit tumor growth. In addition, these phenomena were caused by tumor infiltrated macrophages and they altered these phenotypes. In this review, we will describe our findings on the mechanism of tumor growth suppression by HIF-PH inhibitors. We also want to mention the risks and benefits of future HIF-PH inhibitors.

CD47-mediated immune evasion in early-stage lung cancer progression

Alveolar and interstitial macrophages play crucial roles in eradicating pathogens and transformed cells in the lungs. The immune checkpoint CD47, found on normal and malignant cells, interacts with the SIRPα ligand on macrophages, inhibiting phagocytosis, antigen presentation, and promoting immune evasion. In this study, we demonstrated that CD47 is not only a transmembrane protein, but that it is also highly concentrated in extracellular vesicles from lung cancer cell lines and patient plasma. Abundant CD47 was observed in the cytoplasm of lung cancer cells, aligning with our finding that it was packed into extracellular vesicles for physiological and pathological functions. In our clinical cohort, extracellular vesicle CD47 was significantly higher in the patients with early-stage lung cancer, emphasizing innate immunity inactivation in early tumor progression. To validate our hypothesis, we established an orthotopic xenograft model mimicking lung cancer development, which showed increased serum soluble CD47 and elevated IL-10/TNF-α ratio, indicating an immune-suppressive tumor microenvironment. CD47 expression led to reduced tumor-infiltrating macrophages during progression, while there was a post-xenograft increase in tumor-associated macrophages. In conclusion, CD47 is pivotal in early lung cancer progression, with soluble CD47 emerging as a key pathological effector.

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.

Predictive and Prognostic Relevance of Tumor-Infiltrating Immune Cells: Tailoring Personalized Treatments against Different Cancer Types.

TIICs are critical components of the TME and are used to estimate prognostic and treatment responses in many malignancies. TIICs in the tumor microenvironment are assessed and quantified by categorizing immune cells into three subtypes: CD66b+ tumor-associated neutrophils (TANs), FoxP3+ regulatory T cells (Tregs), and CD163+ tumor-associated macrophages (TAMs). In addition, many cancers have tumor-infiltrating M1 and M2 macrophages, neutrophils (Neu), CD4+ T cells (T-helper), CD8+ T cells (T-cytotoxic), eosinophils, and mast cells. A variety of clinical treatments have linked tumor immune cell infiltration (ICI) to immunotherapy receptivity and prognosis. To improve the therapeutic effectiveness of immune-modulating drugs in a wider cancer patient population, immune cells and their interactions in the TME must be better understood. This study examines the clinicopathological effects of TIICs in overcoming tumor-mediated immunosuppression to boost antitumor immune responses and improve cancer prognosis. We successfully analyzed the predictive and prognostic usefulness of TIICs alongside TMB and ICI scores to identify cancer's varied immune landscapes. Traditionally, immune cell infiltration was quantified using flow cytometry, immunohistochemistry, gene set enrichment analysis (GSEA), CIBERSORT, ESTIMATE, and other platforms that use integrated immune gene sets from previously published studies. We have also thoroughly examined traditional limitations and newly created unsupervised clustering and deconvolution techniques (SpatialVizScore and ProTICS). These methods predict patient outcomes and treatment responses better. These models may also identify individuals who may benefit more from adjuvant or neoadjuvant treatment. Overall, we think that the significant contribution of TIICs in cancer will greatly benefit postoperative follow-up, therapy, interventions, and informed choices on customized cancer medicines.

Has-miR-199a-3p/RELA/SCD inhibits immune checkpoints in AMD and promotes macrophage-mediated inflammation and pathological angiogenesis through lipid metabolism pathway: A computational analysis.

More and more evidence shows that abnormal lipid metabolism leads to immune system dysfunction in AMD and promotes the occurrence of AMD by changing the homeostasis of ocular inflammation. However, the molecular mechanism underlying the effect of lipid metabolism on the phenotype and function of macrophages is still unclear, and the mechanism of association between AMD and cancer and COVID-19 has not been reported. The purpose of this study is to explore the interaction between lipid metabolism related genes, ferroptosis related genes and immunity in AMD, find out the key genes that affect the ferroptosis of AMD through lipid metabolism pathway and the molecular mechanism that mediates the action of macrophages, and find out the possible mechanism of lipid metabolism and potential co-therapeutic targets between AMD and cancer and COVID-19, so as to improve treatment decision-making and clinical results. For the first time, we have comprehensively analyzed the fatty acid molecule related genes, ferroptosis related genes and immune microenvironment of AMD patients, and determined that mast cells and M1 macrophages are the main causes of AMD inflammation, and found that SCD is the core gene in AMD that inhibits ferroptosis through lipid metabolism pathway, and verified the difference in the expression of SCD in AMD in a separate external data set. Based on the analysis of the mechanism of action of the SCD gene, we found for the first time that Has-miR-199a-3p/RELA/SCD is the core axis of action of lipid metabolism pathway to inhibit the ferroptosis of AMD. By inhibiting the immune checkpoint, we can enhance the immune cell activity of AMD and lead to the transformation of macrophages from M2 to M1, thereby promoting the inflammation and pathological angiogenesis of AMD. At the same time, we found that ACOX2 and PECR, as genes for fatty acid metabolism, may regulate the expression of SCD during the occurrence and development of COVID-19, thus affecting the occurrence and development of AMD. We found that FASD1 may be a key gene for the joint action of AMD and COVID-19, and SCD regulates the immune infiltration of macrophages in glioma and germ line tumors. In conclusion, our results can provide theoretical basis for the pathogenesis of AMD, help guide the treatment of AMD patients and their potentially related diseases and help to design effective drug targets.

Abstract LB338: Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma

Abstract Background: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. Methods: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. Results: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. Conclusion: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients. Citation Format: Jeongsoo Kim, Tae-Jin Kim, Sehyun Chae, Hyojeong Ha, Yejin Park, Seon Ah Lim, Hyemin Lee, Younji Sim, Seyoung Oh, Geon Mo Koo, Minkyung Jo, Daehee Hwang, Kyung-Mi Lee. Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB338.

233 Evolutionary Analysis Identifies Early Responsive Macrophage Representing as Anti-tumor Myeloid Cell in Glioblastoma

INTRODUCTION: Glioblastoma (GBM) has no effective treatments. Although immunotherapy has shown promise in certain cancer types, it has not been effective against GBM, largely due to its highly immunosuppressive tumor microenvironment (TMEs), which is rich in tumor-associated macrophages/microglia (TAMs). TAMs in late-stage GBM contribute to T-cell exhaustion and worsen prognosis, but the role of TAMs in earlier stages of tumor development is unclear. METHODS: By employing genetically engineered mouse models and human samples, we used spatiotemporal single-cell transcriptomics to investigate TAM evolution during GBM progression. RESULTS: We identified a previously unknown macrophage state, termed early responsive macrophage (ERM), that exhibited antitumoral activity. ERMs were found to be abundant in the early stages of GBM progression and spatially enriched in the invading edges of GBMs. ERMs are characterized by upregulated immune response signatures, particularly pathways related to antigen processing and presentation, as well as genes associated with phagocytosis and chemotaxis. Mechanistically, we identified QKI, a transcriptional co-activator, as a key player that activates antigen processing and presentation and MHCII expression in ERMs. Loss of QKI in TAMs is associated with the transformation of ERMs into exhausted tumor-infiltrating macrophages (TIMs), which promote an immune-suppressive TME. Therapeutically, enhancement of QKI's transcriptional co-factor activity greatly improved the efficacy of immune checkpoint blockade. CONCLUSIONS: Our study sheds light on the evolutionary trajectory of TAMs in GBM and presents a novel therapeutic approach to enhance their antitumoral activity, thereby constraining tumor growth.

Characterization of Immune Cell Heterogeneity, Key B Cell Subtypes, and Macrophages Functions Using Multiplex Immunofluorescence and Single-cell RNA Sequencing in Chordomas

INTRODUCTION: Chordomas are rare tumors that arise from remnants of the notochord. The standard of care for chordoma remains only surgery and radiotherapy. Though checkpoint inhibition therapy shows promise, recent immunotherapy trials failed to reach endpoints. This emphasizes the need to comprehensively understand the tumor and peripheral immune landscape of chordomas to establish a rationale for immunotherapeutic intervention. METHODS: We analyzed and quantified the immune cell composition (CD3+, CD4+, CD8+, CD20+, and CD163+ cells) of 33 chordomas (14 clival, 3 cervical, 2 lumbar, and 14 sacral tumors) through multiplex immunofluorescence. In addition, we investigated the phenotype of tumor-infiltrating and peripheral B cells and myeloid cells from five chordoma patients employing single-cell RNA sequencing. RESULTS: Hierarchical clustering analysis of 33 chordomas identified 3 distinct clusters based on the relative abundance of B cells, T cells, and macrophages. Cluster 1 was characterized by moderate immune cell infiltration, cluster 2 was immune-desertic, and cluster 3 was highly infiltrated by B and T cells. Findings from single-cell transcriptomics revealed that regulatory B cells and plasma cells were the most abundant B cell populations infiltrating tumors. In addition, we found the presence of tumor-infiltrating macrophages with angiogenic and complement-related functions. CONCLUSIONS: Our study provides crucial insights into the immune landscape of chordomas. The identification of distinct clusters based on immune cell infiltration emphasized the heterogeneity of chordomas. The findings from this study may serve as a foundation for future research and the development of targeted immunotherapies for chordomas.

Abstract 165: PVRL2 upregulation by tumor-associated macrophages confers a regulatory phenotype and is associated with poor prognosis in high-grade serous ovarian cancer

Abstract Advanced or recurrent high-grade serous ovarian cancer (HGSOC) are associated with poor prognosis. While HGSOC is moderately immunogenic, anti-PD-1/L1 therapy has fared poorly in trials. PVRL2 (Nectin2) is both a cell-adhesion molecule as well as a ligand for PVRIG, a novel co-inhibitory receptor. While PVRL2 is constitutively expressed by epithelial/tumor cells, it can also be upregulated by macrophages. Whether macrophage-expressed PVRL2 functions exclusively as an immunoregulatory ligand in HGSOC is not known. CD45+ tumor infiltrate from 6 treatment-naïve and 3 neoadjuvant chemotherapy-treated patients with HGSOC was evaluated by CITE-seq. Multiplex immunofluorescence (mIF) of PanCK, CD68 and PVRL2 was performed on 28 FFPE sections. In vivo, we monitored survival of wild-type and PVRL2−/− mice as well as corresponding bone marrow chimeras orthotopically implanted with ID8-Vegf-Defb29 ovarian tumors. CITE-seq was run on CD45+ cells sorted from wild-type vs PVRL2−/− tumors and ascites. In tumor-bearing mice, macrophages, CD8 or NK cells were depleted and differences in survival assessed. In vitro, M1 and M2 macrophages were differentiated from steady-state bone marrow progenitors and myeloid gene profiles were analyzed. CITE-seq of patient-derived immune cells revealed that PVRL2 expression was restricted to macrophage clusters. Macrophages with low PVRL2 expression showed M1 polarity and abundant expression of inflammatory cytokines and chemokines. Overall macrophage counts and PVRL2 expression were lower in post-chemotherapy cases. In mIF, PVRL2 staining was observed on CD68-positive cells as well as tumor cells. In addition, PVRL2-expressing macrophages accumulated in tumor areas, especially in residual tumor areas after chemotherapy. PVRL2−/− mice and PVRL2 bone marrow chimeras had significantly improved survival compared to their respective control cohorts. Macrophage depletion significantly shortened the prognosis of PVRL2−/− mice compared to wild-type mice. In contrast, depletion of CD8 T cells or NK cells had no effect on survival of wild-type and PVRL2−/− mice. M1 macrophages from PVRL2−/− mice expressed higher levels of inflammatory cytokines and chemokines compared to their wild-type counterparts. Furthermore, CITE-seq showed that tumor-infiltrating macrophages in PVRL2−/− mice exhibited M1 polarization compared to those from wild-type tumors. However, the inflammatory phenotype appeared to be linked more with the tumor-infiltrating macrophages than with ascites macrophages. In conclusion, our research suggests that PVRL2 upregulation by tumor-infiltrating macrophages polarizes towards a pro-tumor phenotype and affects prognosis of HGSOC. PVRL2-deficient macrophages exert their antitumor effects via inflammatory tumor killing and not via antigen-presentation to effector T cells. Citation Format: Kosuke Murakami, Hirofumi Ando, Michele Doucet, Dacheng Ding, William Huang, Sudipto Ganguly. PVRL2 upregulation by tumor-associated macrophages confers a regulatory phenotype and is associated with poor prognosis in high-grade serous 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 165.

Abstract 7048: Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages

Abstract Recent epidemiological data suggests a rising incidence of breast angiosarcoma (AS-B) in the Western population, with over two-thirds related to irradiation or chronic lymphedema. However, unlike head and neck angiosarcoma (AS-HN), AS-B disease characteristics in Asia remains unclear. We examined clinical patterns of angiosarcoma patients (n = 176) seen in an Asian tertiary cancer centre from 1999 to 2021, and specifically investigated the molecular and immune features of AS-B in comparison to AS-HN. Data from whole genome sequencing (WGS) (AS-HN, n = 44; breast, n = 7), NanoString PanCancer IO360 gene expression profiling (AS-HN, n = 42; breast, n = 7) and 10X Visium spatial transcriptomics (AS-HN, n = 4; breast, n = 2) were analyzed. Majority of cases were AS-HN (n = 104; 59.1%), while AS-B (n = 16, all females) accounted for 9.1% of the cases. The median age at diagnosis was 43 years (range, 26 to 74). Four cases (25%) were related to prior irradiation, while 6 out of 8 cases evaluated were positive for human herpesvirus-7 on immunohistochemistry. Although all cases were non-metastatic at diagnosis and were treated with curative-intent surgery, 6 patients (37.5%) experienced rapid relapse within a median of 7.8 months. Based on WGS, 4 of the 7 AS-B had non-synonymous somatic variants in 48 genes (range, 2 to 28 per case). Among the notable variants found were in kinases (KDR, JAK2, FLT4, ATR, EPHB1 and PRKACA). Other variants were found in cancer-related genes (e.g. ATRX, FOXO1, SMAD3, PTCH1, NOTCH4, HIF1A and SETD1A). These genes were functionally annotated by performing over representation analysis (www.webgestalt.org) on publicly available databases (e.g. Gene Ontology and KEGG). They were enriched in cancer-related pathways such as regulation of cell differentiation, VEGFR and receptor tyrosine kinase signaling pathways. By NanoString profiling, compared to AS-HN, AS-B was enriched for angiogenesis, notch signaling and metastasis-associated matrix remodeling pathways. Conversely, AS-B was depleted of lymphoid compartment, immune cell adhesion and migration expression signatures. Based on the gene expression signatures, AS-B was enriched for macrophages, CD8+ T cells and was depleted for T-reg cells. Furthermore, spatial transcriptomics showed that compared to AS-HN, tumor-infiltrating leukocytes (TILs) in AS-B were enriched for macrophage and T-cells but were depleted for B-cells and NK cells. The location of TILs could provide information on the feasibility of immunotherapy in angiosarcoma. In conclusion, we observed a convergence of both mutational and expression signatures on angiogenesis signaling pathways in our AS-B cases. The spatial transciptomic data revealed that AS-B was enriched for tumor-infiltrating macrophages and T-cells which might have implications on the role of immunotherapy. Citation Format: Tun Kiat Ko, Zexi Guo, Bavani Kannan, Boon Yee Lim, Jing Yi Lee, Abner Herbert Lim, Zhimei Li, Elizabeth Chun Yong Lee, Cedric Chuan-Young Ng, Bin Tean Teh, Jason Yongsheng Chan. Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages [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 7048.

Abstract 6873: Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor microenvironment

Abstract Squamous cell carcinoma antigen 1 (SCCA1) is a potential biomarker and prognostic indicator for many epithelial cancers including cervix, head and neck, breast, and lung. Cancer patients with high serum levels of SCCA commonly experience treatment resistance and recurrence. We analyze SCCA expression in correlation with tumor microenvironment from 66 human cervical tumor biopsies and find that high serum SCCA is associated with increased macrophage infiltration in tumors and poor overall survival. In a single cell RNAseq analysis of 8 cervical tumors, patients with high SCCA1 show increased immunosuppressive genes (C1QA, C1QC, APOE) and reduced MHC molecules (HLA-DR, HLA-DQA1) in their tumor associated macrophages (TAMs), corresponding to decreased expression of T-cell cytotoxic-associated genes (IFNG, GZMB, PRF1). Thus, we aim to understand the impact of elevated SCCA1 on macrophages and its role in shaping the immune response. Co-culture of peritoneal macrophages isolated from SCCA1-treated mice with T cells from syngeneic wild-type mice, showed significant suppression in T cell proliferation. Downregulation of antigen presentation molecules was also observed SCCA1-treated macrophages and resulted in impaired ability to induce T cell activation by peptide-pulsed macrophages. Compared with treatment-naïve mice, SCCA1-treated macrophages showed increased anti-inflammatory cytokine CCL22, IL10 and immunosuppression regulators ARG1, PD-L1 expression. Moreover, human monocyte-derived M2 macrophages treated with SCCA1 during the differentiation process demonstrated inhibition toward CD3/28-induced T cell proliferation, accompanied by enhanced expression of cytokines, IL10 and CCL18. We further found that targeting SCCA1 by small interfering RNA knockdown significantly reduced tumor growth and macrophage infiltration, resulting in improved T-cell antitumor immunity. Lastly, analysis of cervical cancer patients with chemoradiation therapy showed that patients with high SCCA are associated with increased exhaustion (LAG3, CTLA4) and decreased antitumor gene signature (GZMB, IFNG) during treatment. In summary, SCCA expression promotes immunosuppressive macrophage polarization and function, contributing to T cell exhaustion phenotype and poor treatment response. Citation Format: Liyun Chen, Victoria Shi, Eric Liu, Jasmine Yang, Abhay Singh, Matthew Inkman, Jin Zhang, Julie Schwarz, Stephanie Markovina. Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor 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 6873.

Abstract 7458: Inhibition of brain cancer progression via resolution of inflammation

Abstract While inflammation is a hallmark of cancer, the mechanisms that disrupt its resolution in cancer are not well understood. A paradigm shift is emerging in our understanding that the resolution of inflammation is an active process controlled by specialized pro-resolving mediators (SPMs), including protectins and resolvins, which are endogenous lipid autacoids present in multiple tissues including the brain and cerebrospinal fluid. SPMs counter-regulate pro-inflammatory cytokine production and stimulate phagocytosis of cellular debris (“pro-resolution”) without being immunosuppressive. We hypothesized treatment with pro-resolving agonists such as SPMs presents a novel debris-clearing and resolution-enhancing therapeutic approach to complement cytotoxic brain cancer therapies. Here, we demonstrate the failure of resolution of inflammation via dysregulated SPMs may be a critical risk factor for aggressive brain cancer growth. Moreover, chemotherapy and targeted therapy further disrupt inflammation resolution via a tumor cell death (“debris”)-induced cytokine storm by macrophages. The SPM receptors GPR37 (protectin D1), GPR18/DRV2 (resolvin (Rv) D2), GPR32 (RvD1), and ChemR23/ERV (RvE1) are expressed in tumor-infiltrating macrophages and endothelial cells in the brain tumor microenvironment. Plasma levels of SPMs were significantly reduced in the medulloblastoma-bearing mice, suggesting that brain cancer progression is associated with a failure of endogenous resolution of inflammation. To determine whether failed resolution in brain cancer can be rescued by lipid mediator agonists, we systemically administered SPMs to subcutaneous and orthotopic glioblastoma and medulloblastoma murine brain tumor models. Significantly, RvD1 and RvD2 each inhibited the growth of human glioblastoma tumors by 15-fold at nanogram concentrations without toxicity. Likewise, RvD2 inhibited the growth of human medulloblastoma tumors by 3-fold. Protectins, PDX and PD1n-3, each inhibited growth of human medulloblastoma in mice by approximately 5-fold. Systemic administration of PDX or RvD2 prolonged survival of mice injected with orthotopic human D556 medulloblastoma and also glioblastoma models. Flow cytometry analysis of orthotopic glioblastoma tumors revealed increased immune infiltration into the brain tumor microenvironment in the PCTR1-treatment group when compared to the tumor-bearing non-treated group. At nanogram per day doses, protectins and resolvins suppressed orthotopic debris-stimulated brain tumor growth by activating macrophage and microglial phagocytosis of tumor cell debris and counter-regulating pro-inflammatory cytokines, including TNF-α, CCL2, CXCL1, and CCL4. Thus, stimulating the resolution of inflammation via the clearance of cellular debris with pro-resolving agonists represents a novel therapeutic approach to brain cancer, including glioblastoma and medulloblastoma. Citation Format: Jacqueline Capuano, Kimberly Vasquez, Katherine Quinlivan, Rachel Bayer, Sarina Virani, Michael Gillespie, Mark Kieran, Charles Serhan, Diane Bielenberg, Dipak Panigrahy. Inhibition of brain cancer progression via resolution of inflammation [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 7458.

Abstract 1522: Tumor-associated fibrosis leads to an increase in T-cell exhaustion and impairs tumor control in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related mortality with non-small cell lung cancer (NSCLC) being the most predominant type. Recent studies have shown the efficacy of anti-PD1 inhibitors which improved overall survival. However, this efficacy is limited to a subset of patients due to the intrinsic tumor resistance to immune checkpoint inhibitors. Our group recently demonstrated that tumor-associated fibrosis negatively correlates with T cell-mediated immune response in lung cancer. This is largely dependent on Col13a1-expressing cancer-associated fibroblasts (CAFs), which result in recruiting the immunosuppressive macrophages and regulatory T cells while limiting dendritic cells and CD8 T cell infiltration. Here, we characterize the effect of fibrosis on the TME including TILs and macrophages using an orthotopic KRAS and P53-driven murine NSCLC model (KPL86-mCher model) with FITC-induced fibrosis.Fibrosis significantly increased the number of cancer-associated fibroblasts and collagen density in KPL86-mCher lung tumors. Following carboplatin + pemetrexed and Anti-PD1 treatment, we observed no increase in tumor-infiltrating CD4+ and CD8+ T cells or decrease in tumor burden when compared with the non-fibrotic tumors. Rather, we observed a significant increase in exhausted T cells marked by high Tim3, PD1, and TOX in fibrotic KPL86-mCher tumors. Corresponding to these impaired T-cell responses, we found an increase in tumor-infiltrating alveolar macrophages with immunosuppressive phenotype under fibrotic tumor conditions. Our data suggests that tumor-associated fibrosis leads to an increase in CD8+ T cell exhaustion as well as immunosuppressive macrophage infiltration in our spontaneously derived murine model on NSCLC. Overall, this indicates that targeting tumor fibrosis may significantly improve the efficacy of immunotherapy which would have translational implications for patients with immunotherapy-resistant NSCLC. Citation Format: Usman Y. Panni, Varun Shenoy, Brett Knolhoff, Faiz Ahmed, Brett Herzog, William G. Hawkins, John A. Olson, David G. DeNardo. Tumor-associated fibrosis leads to an increase in T-cell exhaustion and impairs tumor control 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 1522.

Abstract 1538: Impact of C-reactive protein on the efficacy of immune checkpoint inhibitors in non-small cell lung cancer

Abstract Background: Immune checkpoint inhibitors (ICIs) therapy has achieved remarkable success in the treatment of non-small cell lung cancer (NSCLC). Clinical studies have suggested a correlation between C-reactive protein (CRP) levels and treatment efficacy, but it remains unclear whether CRP can directly impact the efficacy. Therefore, we investigated the imapct of CRP on the efficacy of ICIs in NSCLC. Methods: The tumor growth model was established in CRP knockout and wildtype mice by injecting LLC cells to evaluate the effect of CRP on tumor proliferation. The frequencies and phenotypes of macrophages and T cells in mouse tumors were detected by flow cytometry. The impact of CRP on macrophage polarization was evaluated using flow cytometry and IF, while its impact on T cell chemokines CCL5, CXCL9, and CXCL10 was analyzed by ELISA and qRT-PCR. mCRP and α-PD-1 were implemented in C57/BL6 mice injected with LLC cells to evaluate the effect of mCRP on tumor immunotherapy. The frequencies of T cells in mouse tumors were detected by flow cytometry. Expression of mCRP and infiltration of immune cells in human NSCLC tissues were analyzed using IHC. Results: We found that tumor progression was enhanced and the proportion of tumor-infiltrated M1 macrophages was decreased in CRP knockout mice. Using human PBMC-derived macrophage and RAW264.7 cell lines, we demonstrated that the monomeric form of CRP, known as mCRP, induced M1 polarization of macrophages, leading to increased secretion of CCL5, CXCL9, and CXCL10. In tumor bearing mice, mCRP treatment enhanced anti-tumor immunity and suppressed tumor progression by increasing CD4+ and CD8+ T cell recruitment. Surprisingly, the combined treatment of mCRP and α-PD-1 induced more significant tumor regression than α-PD-1 treatment alone. Additionally, high level of mCRP in NSCLC tissues is correlated with a more inflamed tumor microenvironment phenotype. Conclusion: mCRP can enhance the efficacy of immune checkpoint inhibitors in NSCLC by activating anti-tumor immunity. Citation Format: Wenyuan Li, Hui Guo. Impact of C-reactive protein on the efficacy of immune checkpoint inhibitors 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 1538.

Abstract 2662: Deciphering the cellular and molecular determinants of immunotherapy resistance in NASH-associated hepatocellular carcinoma by single-cell analysis

Abstract Introduction: Non-alcoholic steatohepatitis (NASH) has emerged as a prominent risk and the most rapidly increasing aetiology of hepatocellular carcinoma (HCC). Despite the effectiveness of immune checkpoint blockade (ICB), like anti-programmed cell death-ligand 1 (anti-PD-L1) antibodies, in a subset of HCC patients, NASH has been shown to impair the anti-tumor immunity in ICB-treated HCC. We aim to elucidate the mechanisms responsible for ICB resistance in NASH-HCC at single-cell resolution. Methods: Orthotopic NASH-HCC mouse models were generated by orthotopically injecting liver cancer cell line into NASH liver induced by methionine-and choline-deficient diet (MCD) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Multi-color flow cytometry and single-cell RNA-sequencing (scRNA-seq) were utilized to dissect the liver and tumor immune microenvironment upon anti-PD-L1 treatment. Results: Anti-PD-L1 treatment failed to alleviate tumor burden in NASH mice, indicating that orthotopic NASH-HCC mouse models were resistant to ICB therapy. Compared to the control liver, multi-color flow cytometry demonstrated that CD11b+F4/80+CD206+M2 macrophages accumulated in NASH liver and further increased in liver and tumor upon anti-PD-L1 treatment. Tumor-infiltrating M2 macrophages exhibited the most significant and robust correlation with tumor weight among all immune cell types. Moreover, PD-1 expression on tumor-infiltrating CD8+ T cells positively correlated with tumor weight and intratumoral M2 macrophages. Furthermore, scRNA-seq analysis identified a NASH-specific tumor-infiltrating monocyte subcluster, which may differentiate into an ‘M2-like’ macrophage subtype upon anti-PD-L1 treatment as demonstrated by cell trajectory analysis. Conclusion: Our study provides insights into the significance of monocyte/macrophage reprogramming in ICB resistance in NASH-HCC. Further investigation is conducted to identify the molecular mechanisms during monocyte-macrophage differentiation and explore potential targeted strategies to overcome ICB resistance in NASH-HCC. Acknowledgment: This study is supported by the General Research Fund (14120621 and 14119023), the Collaborative Research Fund (C4045-18W), the Li Ka Shing Foundation and CUHK Strategic Seed Funding for Collaborative Research Scheme. Keywords: hepatocellular carcinoma, single-cell RNA-sequencing, anti-PD-L1, macrophages. Citation Format: Lingyun Zhang, Yiling Zhang, Wenshu Tang, Zhewen Xiong, Xiaoyu Liu, Zhixian Liang, Yuk Wah Tsang, Weiqin Yang, Xiaohang Long, Alfred Sze Lok Cheng. Deciphering the cellular and molecular determinants of immunotherapy resistance in NASH-associated hepatocellular carcinoma by single-cell analysis [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 2662.

Abstract 289: Temporal and spatial dynamics of glial cells apolipoprotein E expression in astrocytoma microenvironment

Abstract Introduction: Brain tumors have proven challenging to treat due to the complex and sensitive architecture of the brain. Previous research has shown that GBM cells depend highly on cholesterol uptake, and apolipoprotein E (APOE) is required to transport cholesterol among tissues or cells. APOE is primarily synthesized by astrocytes and has been extensively studied in neurodegenerative diseases. While extensively studied in neurodegenerative diseases, limited studies are focusing on their roles in brain tumor development. Here, we analyzed the temporal and spatial expression of APOE during brain tumor progression in a murine astrocytoma tumor model. Material and method: ALTS1C1 astrocytoma cells were intracranially injected into the mouse brain. The animal was sacrificed, and the left hemisphere, right hemisphere, and tumor tissue were collected for flow cytometry analysis. CD45, CD11b, and GFAP were used to recognize macrophages, microglia, and astrocytes. CD68, TMEM119, and GFAP were used for tissue immunostaining to label macrophages, microglia, and astrocytes, respectively. Results and Discussion: Flow cytometry analysis revealed upregulated APOE in tumor-infiltrating microglia, macrophages, and lymphocytes. GFAPhigh astrocytes displayed lower APOE levels than GFAPlow astrocytes. IHC analysis showed that CD68+ macrophages exhibited higher APOE expression at the tumor edge than normal tissue. On the contrary, GFAP+ astrocytes at the tumor edge displayed a lower APOE signal than the normal tissue. Previous studies have shown the participation of APOE in regulating immune response in various cancers. Further studies to analyze the dynamic change of APOE on the gene expression level with in situ hybridization and the quantification of cholesterol levels in the microenvironment may help uncover their roles in the glioma microenvironment. Conclusion: This study demonstrated that APOE was temporal and spatial differentially expressed in macrophages and astrocytes in the brain tumor microenvironment. Tumor-infiltrating cells showed an elevated APOE expression, while GFAP+ astrocytes expressed reduced APOE levels. Although the mechanism of these alterations remains unclear, this study provides a basis for future studies about the roles of APOE in the brain tumor microenvironment. Citation Format: Ting-Yi Chien, Chi-Shiun Chiang. Temporal and spatial dynamics of glial cells apolipoprotein E expression in astrocytoma 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 289.

Abstract 4187: Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Abstract Background: Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human cancer and immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs, including immune checkpoint inhibitors (ICIs), which have demonstrated limited effectiveness in OC patients. Methods: We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). We assessed the efficacy of PD-1 blockade with pembrolizumab in the SKOV-3 Luc Hu-mouse model. The immune profiles of the tumors were characterized using colorimetric immunostaining and flow cytometry. Additionally, we analyzed RNA-seq data to investigate the gene expression signature of pembrolizumab refractory tumors. Results: We confirmed tumor development in both OC cell lines within CD34+ Hu-mice. In these models, human lymphocyte and myeloid cell subsets were present in the tumors, draining lymph nodes, blood, and spleens. The SKOV-3 Luc tumor-bearing Hu-mice did not respond to pembrolizumab monotherapy. These tumors exhibited a high presence of tumor-infiltrating macrophages. Tumors in Hu-mice unresponsive to pembrolizumab showed a lower abundance of CD8+ T-cells, memory B cells, plasma cells, and a higher proportion of naïve M0 macrophages and mast cells compared to the PBS control. Furthermore, we identified 43 significantly enriched gene sets in these tumors. The differentially expressed genes (DEGs) were predominantly enriched in HDAC class I, RB1, KLF1/3, TCF21, MYD88, SMARCE1 target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts. Conclusion: Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer potential avenues to explore mechanisms of resistance to PD-1/PD-L1 blockade in OC. Citation Format: Sung Wan Kang, Ji-young Lee, Ok-Ju Kang, Yong-Man Kim, Eun Kyung Choi, Shin-Wha Lee. Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice [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 4187.