Tumor-infiltrating Macrophages Research Articles

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

IntroductionRecent 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 remain unclear.MethodsWe examined clinical patterns of angiosarcoma patients (n = 176) seen in an Asiantertiary cancer center 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), NanoString gene expression profiling and 10x Genomics Visium spatial transcriptomics were analyzed.ResultsMajority 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). Based on WGS, 4 of the 7 AS-B had non-synonymous somatic variants in 47 genes (range, 2 to 28 per case). These genes were functionally annotated and were enriched in cancer-related pathways such as regulation of cell differentiation, VEGFR and receptor tyrosine kinases signaling pathways. By NanoString gene expression profiling, ASB, compared to AS-HN, were enriched for angiogenesis, notch signaling and metastasis-associated matrix remodeling pathways. Additionally, AS-B were enriched for macrophages and CD8+ T cells expression signatures. Similarly, Visium spatial transcriptomics showed that AS-B were enriched for macrophages and T-cells.DiscussionIn conclusion, in our AS-B cases, we observed a convergence of both mutational and expression signatures on angiogenic-related pathways. Thus, anti-angiogenic therapy could be an option to treat AS-B.

Computational pathology applied to clinical colorectal cancer cohorts identifies immune and endothelial cell spatial patterns predictive of outcome.

Colorectal cancer (CRC) is a histologically heterogeneous disease with variable clinical outcome. The role the tumour microenvironment (TME) plays in determining tumour progression is complex and not fully understood. To improve our understanding, it is critical that the TME is studied systematically within clinically annotated patient cohorts with long-term follow-up. Here we studied the TME in three clinical cohorts of metastatic CRC with diverse molecular subtype and treatment history. The MISSONI cohort included cases with microsatellite instability that received immunotherapy (n = 59, 24 months median follow-up). The BRAF cohort included BRAF V600E mutant microsatellite stable (MSS) cancers (n = 141, 24 months median follow-up). The VALENTINO cohort included RAS/RAF WT MSS cases who received chemotherapy and anti-EGFR therapy (n = 175, 32 months median follow-up). Using a Deep learning cell classifier, trained upon >38,000 pathologist annotations, to detect eight cell types within H&E-stained sections of CRC, we quantified the spatial tissue organisation and colocalisation of cell types across these cohorts. We found that the ratio of infiltrating endothelial cells to cancer cells, a possible marker of vascular invasion, was an independent predictor of progression-free survival (PFS) in the BRAF+MISSONI cohort (p = 0.033, HR = 1.44, CI = 1.029-2.01). In the VALENTINO cohort, this pattern was also an independent PFS predictor in TP53 mutant patients (p = 0.009, HR = 0.59, CI = 0.40-0.88). Tumour-infiltrating lymphocytes were an independent predictor of PFS in BRAF+MISSONI (p = 0.016, HR = 0.36, CI = 0.153-0.83). Elevated tumour-infiltrating macrophages were predictive of improved PFS in the MISSONI cohort (p = 0.031). We validated our cell classification using highly multiplexed immunofluorescence for 17 markers applied to the same sections that were analysed by the classifier (n = 26 cases). These findings uncovered important microenvironmental factors that underpin treatment response across and within CRC molecular subtypes, while providing an atlas of the distribution of 180 million cells in 375 clinically annotated CRC patients. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Chemokine (C-C Motif) Ligand 2/CCR2/Extracellular Signal-Regulated Kinase Signal Induced through Cancer Cell-Macrophage Interaction Contributes to Hepatocellular Carcinoma Progression.

Tumor-infiltrating macrophages, known as tumor-associated macrophages, play a crucial role in the tumor microenvironment. Immunohistochemistry revealed that intratumoral CD68-positive macrophages are associated with poor prognosis and clinicopathologic factors in patients with hepatocellular carcinoma (HCC). Subsequently, an indirect co-culture system involving HCC cells and peripheral blood-derived macrophages was developed. cDNA microarray analysis revealed that chemokine (C-C motif) ligand 2 (CCL2) was highly expressed in HCC cells co-cultured with macrophages. CCL2 neutralization suppressed proliferation, migration, and phosphorylation of extracellular signal-regulated kinase (Erk) in HCC cells and macrophages enhanced through co-culture. In contrast, recombinant human CCL2 (rhCCL2) addition facilitated these malignant phenotypes and increased Erk phosphorylation levels in HCC cells and macrophages. The primary CCL2 receptor, CCR2, was expressed in HCC cells and macrophages and was up-regulated in co-cultured HCC cells. CCR2 inhibition suppressed malignant phenotypes and reduced phosphorylated levels of Erk enhanced by rhCCL2. Additionally, the inhibition of Erk signal suppressed rhCCL2-enhanced malignant phenotypes. Moreover, serum CCL2 levels were higher in patients with HCC than those in healthy donors. On the basis of immunohistochemistry, CCL2-positive cases with high CCR2 expression and phosphorylated Erk-positive cases exhibited poor survival outcomes. Therefore, CCL2 up-regulation through interactions between HCC cells and macrophages contributed to HCC progression, making the CCL2/CCR2/Erk signal a potential target for HCC treatment.

Nucleotide receptor P2X7/STAT6 pathway regulates macrophage M2 polarization and its application in CAR-T immunotherapy.

A key factor underlying the failure of Chimeric Antigen Receptor-T Cell (CAR-T) therapy in ovarian cancer (OC) is the presence of an immunosuppressive tumor microenvironment, which is intricately linked to M2 polarization among tumor-infiltrating macrophages. P2X7 receptor has been previously documented as expressed within these macrophages and its correlation with M2 polarization is evident. This investigation scrutinizes whether silencing of P2X7 receptor within macrophages could lead to augmented anti-tumor potency of CAR-T. Human peripheral blood mononuclear cells were artificially differentiated into macrophages or M2 macrophage in vitro. After silencing P2X7 receptor and/or overexpressing STAT6 within macrophages, the M1 and M2 markers were evaluated via flow cytometry, ELISA, and qRT-PCR. Additionally, the phosphorylation level of STAT6 was monitored by western blot. We engineered CAR-T cells targeting the non-functional P2X7 (nfP2X7) receptor, and co-cultured them with macrophages silencing P2X7 receptor along with OC cells. The anti-tumor effect of these CAR-T cells was assessed through evaluating OC cell viability, lactate dehydrogenase release, and IFN-γ levels. P2X7 receptor silencing promotes M1 macrophage marker expression (CD86, TNF-α, IL-6, IL-1β), diminishes M2 macrophage marker expression (CD206 and IL-10) and suppresses STAT6 phosphorylation, whereas STAT6 overexpression reverses these phenomena. Furthermore, M2 macrophage suppresses the toxic effect of CAR-T cells on OC cells, while silencing the P2X7 receptor nullifies the immunosuppressive effect of M2 macrophages on CAR-T cells. Silencing P2X7 receptor can reverse M2 macrophage polarization by suppressing STAT6 activation, thereby enhancing the anti-tumor efficacy of CAR-T cells targeting nfP2X7 receptor in OC cell lines.

Prolonged survival by combination treatment with a standardized herbal extract from Japanese Kampo-medicine (Juzentaihoto) and gemcitabine in an orthotopic transplantation pancreatic cancer model.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its poor prognosis. Traditional Japanese herbal medicine (Kampo), such as Juzentaihoto (a standardized combination of 10 herbal extracts), has shown immune modulatory effects, modulation of microcirculation, and amelioration of fatigue. It is administered to patients to prevent deterioration of cachexia and counteract side effects of chemotherapy. The effect of Juzentaihoto with or without standard chemotherapy (Gemcitabine) on survival and tumor microenvironment was studied in an immunocompetent pancreatic cancer mouse model. Following tumor development ±12 days after orthotopic implantation of murine pancreatic cancer cells (KPC) into the pancreas of C57BL/6 mice, the mice were treated with Gemcitabine, Juzentaihoto, their combination (Gem/Juz) or NaCl (Ctr.). Combination treatment significantly prolonged survival (+38%) of tumor bearing mice, compared to controls as well as Gemcitabine or Juzentaihoto monotherapy. Macrophage (CD68+) infiltration in pancreatic tumors was significantly enhanced in Gem/Juz - treated animals, compared with controls (p < 0,001), with significant increases of both, macrophages (CD68+) and for lymphocytes (CD45+), especially at the tumor front. In vitro, Juz- or Gem/Juz-treated KPC tumor cells secreted significantly more macrophage-chemoattractant cytokines, e.g., CCL2, CCL20, and CXCL2, whilst Juz- and Gem/Juz-treated macrophages (MH-S) secreted cytokines of the M1 phenotype, e.g., IL6, TNF-α, and IL12. It has been shown that tumor cells recruit and polarize macrophages towards tumor-associated macrophages (TAM). Our results indicate a change in macrophage polarization which not only induced anti-tumor immune-cell activity and cytokine release, but also suggests amelioration of Gemcitabine efficacy as DNA-analogue and as partial antitumor antigen. We propose that the increased survival of tumor bearing mice after Gem/Juz combination treatment is due to the restored cytotoxicity of Gemcitabine and changes in the tumor-microenvironment - induced by Juzentaihoto - such as an increased number of M1 macrophages.

Comprehensive Multi-Omics Analysis Reveals NPC2 and ITGAV Genes as Potential Prognostic Biomarkers in Gastrointestinal Cancers.

Gastrointestinal cancers (GICs) continue to dominate in terms of both incidence and mortality worldwide. Due to the absence of efficient and accurate prognostic biomarkers, the prognosis and treatment outcomes of many GICs are poor. Identifying biomarkers to predict individual clinical outcomes efficiently is a fundamental challenge in clinical oncology. Although several biomarkers have been continually discovered, their predictive accuracy is relatively modest, and their therapeutic use is restricted. In light of this, the discovery of reliable biomarkers for predicting prognosis and outcome in GIC is urgently required. We evaluated the Human Protein Atlas dataset and identified NPC Intracellular Cholesterol Transporter 2 (NPC2) and Integrin Subunit Alpha V (ITGAV) as probable poor predictive genes for these cancers. In addition, we used the GEPIA2, cBioPortal, UALCAN, LinkedOmics, STRING, Enrichr, TISDB, TIMER2.0, hTFTarget, miRTarBase, circBank, and drug-gene interaction database databases to conduct a comprehensive and systematic analysis of the NPC2 and ITGAV genes. Our results found high expression levels of NPC2 and ITGAV in most GICs. The aforementioned gene expressions were linked to several clinicopathological characteristics of GICs as well as poorer prognosis in LIHC and STAD. The most common alteration type of NPC2 was amplification, and for ITGAV was deep deletion. Significant promotor hypermethylation was also seen in NPC2 and ITGAV in PAAD and COAD, respectively. For the immunologic significance, NPC2 and ITGAV were positively correlated with the abundance of tumor-infiltrating lymphocytes and macrophages. Furthermore, various immunomodulators showed strong correlations with the expression of these genes. There were currently 10 small molecule drugs targeting ITGAV. Consequently, our bioinformatics analysis showed that NPC2 and ITGAV might be used as potential biomarkers to determine the prognosis of various GICs and are also related to immune infiltration.

Cancer cells restrict immunogenicity of retrotransposon expression via distinct mechanisms

To thrive, cancer cells must navigate acute inflammatory signaling accompanying oncogenic transformation, such as via overexpression of repeat elements. We examined the relationship between immunostimulatory repeat expression, tumor evolution, and the tumor-immune microenvironment. Integration of multimodal data from a cohort of pancreatic ductal adenocarcinoma (PDAC) patients revealed expression of specific Alu repeats predicted to form double-stranded RNAs (dsRNAs) and trigger retinoic-acid-inducible gene I (RIG-I)-like-receptor (RLR)-associated type-I interferon (IFN) signaling. Such Alu-derived dsRNAs also anti-correlated with pro-tumorigenic macrophage infiltration in late stage tumors. We defined two complementary pathways whereby PDAC may adapt to such anti-tumorigenic signaling. In mutant TP53 tumors, ORF1p from long interspersed nuclear element (LINE)-1 preferentially binds Alus and decreases their expression, whereas adenosine deaminases acting on RNA 1 (ADAR1) editing primarily reduces dsRNA formation in wild-type TP53 tumors. Depletion of either LINE-1 ORF1p or ADAR1 reduced tumor growth invitro. The fact that tumors utilize multiple pathways to mitigate immunostimulatory repeats implies the stress from their expression is a fundamental phenomenon to which PDAC, and likely other tumors, adapt.

The HDAC6 inhibitor AVS100 (SS208) induces a pro-inflammatory tumor microenvironment and potentiates immunotherapy.

Histone deacetylase 6 (HDAC6) inhibition is associated with an increased pro-inflammatory tumor microenvironment and antitumoral immune responses. Here, we show that the HDAC6 inhibitor AVS100 (SS208) had an antitumoral effect in SM1 melanoma and CT26 colon cancer models and increased the efficacy of anti-programmed cell death protein 1 treatment, leading to complete remission in melanoma and increased response in colon cancer. AVS100 treatment increased pro-inflammatory tumor-infiltrating macrophages and CD8 effector T cells with an inflammatory and T cell effector gene signature. Acquired T cell immunity and long-term protection were evidenced as increased immunodominant T cell clones after AVS100 treatment. Last, AVS100 showed no mutagenicity, toxicity, or adverse effects in preclinical good laboratory practice studies, part of the package that has led to US Food and Drug Administration clearance of an investigational new drug application for initiating clinical trials. This would be a first-in-human combination therapy of pembrolizumab with HDAC6 inhibition for locally advanced or metastatic solid tumors.

Synthesis and preclinical evaluation of [18F]AlF-NODA-MP-C6-CTHRSSVVC as a PET tracer for CD163-positive tumor-infiltrating macrophages

Positron emission tomography (PET) can provide information about tumor-associated macrophage (TAM) infiltration, as long as a suitable tracer is available. This study aimed to evaluate the radiolabeled peptide [18F]AlF-NODA-MP-C6-CTHRSSVVC as a potential PET tracer for imaging of the CD163 receptor, which is expressed on M2-type tumor-associated macrophages. The conjugated peptide NODA-MP-C6-CTHRSSVVC was labeled with aluminum [18F]fluoride. Tracer binding and its biodistribution were evaluated in an in vitro binding assay and in healthy BALB/c mice, respectively. In addition, different treatments with cyclophosphamide in tumor-bearing mice were used to assess whether the tracer could detect differences in CD163 expression caused by differential TAM infiltration. After 7 days of treatment, animals were injected with [18F]AlF-NODA-MP-C6-CTHRSSVVC, and a 60-min dynamic PET scan was performed, followed by an ex vivo biodistribution study. [18F]AlF-NODA-MP-C6-CTHRSSVVC was prepared in 23 ± 6 % radiochemical yield and showed approximately 50 % of specific receptor-mediated binding in an in vitro binding assay on human CD163-expressing tissue homogenates. No CD163-mediated binding of [18F]AlF-NODA-MP-C6-CTHRSSVVC was detected by PET under normal physiological conditions in healthy BALB/c mice. On the other hand, CD163-positive xenograft tumors were clearly visualized with PET and a positive correlation was found between CD163 levels and the [18F]AlF-NODA-MP-C6-CTHRSSVVC tumor-to-muscle ratio (TMR) obtained from the PET images (Pearson r = 0.76, p = 0.002). No significant differences in the CD163 protein level and in the tracer uptake between treatment groups were found in the tumors. Taken together, [18F]AlF-NODA-MP-C6-CTHRSSVVC appears a promising candidate PET tracer for M2-type TAM, as it binds specifically to CD163 in vitro and its tumor uptake correlates well with CD163 expression in vivo.

Selective activation of PPARα by pemafibrate mitigates peritoneal inflammation and fibrosis through suppression of NLRP3 inflammasome and modulation of inflammation

Peritoneal inflammation and fibrosis remain major challenges to the long-term maintenance of peritoneal dialysis. Pemafibrate, a selective peroxisome proliferator-activated receptor α (PPARα) modulator, has been implicated in the management of fibrosis-related disorders. We investigated whether pemafibrate ameliorates peritoneal inflammation and fibrosis and explored the underlying mechanisms in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO mice). MGO mice exhibited peritoneal fibrosis with increased expression of mesenchymal markers, transforming growth factor-β1 (TGF-β1), and substantial deposition of extracellular matrix (ECM) proteins. Additionally, MGO mice exhibited peritoneal inflammation as indicated by elevated tumor necrosis factor-α expression and macrophage infiltration in peritoneal tissue. These effects were mitigated by pemafibrate treatment, which also restored peritoneal membrane function. Furthermore, pemafibrate promoted anti-inflammatory macrophage polarization in both mice and THP-1 cells. In human peritoneal mesothelial cells (HPMCs), pemafibrate effectively inhibited interferon-γ-induced production of TGF-β1 and ECM while suppressing the proinflammatory cytokines nuclear factor-κB (NF-κB) and activator protein 1. The NF-κB inhibitory effect of pemafibrate involved stabilization of the NF-κB inhibitory protein IkBα. Notably, pemafibrate hindered activation of the NLR family pyrin domain containing 3/caspase-1 axis in interferon-γ-stimulated THP-1 cells. These findings suggest that pemafibrate ameliorates peritoneal inflammation and fibrosis, making it a promising candidate for peritoneal fibrosis therapy.

Phase 2 Trial of Regorafenib in Recurrent/Metastatic Adenoid Cystic Carcinoma.

There is a significant need for effective therapies to treat recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC). This study evaluated the multitargeted VEGFR tyrosine kinase inhibitor (TKI) regorafenib in patients with R/M ACC. Patients with progressive R/M ACC were treated with regorafenib until disease progression, consent withdrawal, or excessive toxicity. The co-primary endpoints were best overall response and 6-month progression-free survival (PFS). Genomic and transcriptomic biomarker analyses were performed in tumors from trial participants. Thirty-eight patients were enrolled, including 7 (18%) patients with prior VEGFR TKIs. No objective responses were observed. The 6-month PFS was 45%, and the median PFS was 7.2 months (95% confidence interval, 5.2-11.9 months). The presence of either activating NOTCH1 (22%) or KDM6A alterations (24%) was associated with decreased PFS [HR 2.6; 95% confidence interval (CI), 1.1-6.1; P = 0.03]. Bulk RNA sequencing of pretreatment tumors revealed that regorafenib clinical benefit (CB; PFS ≥ 6 months; n = 11) was associated with the native enrichment of immune-related signatures. Immune deconvolution revealed a greater degree of macrophage and T-cell infiltration in CB tumors. Tumors from patients with no clinical benefit (NCB; PFS < 6 months; n = 9) had greater expression of signatures related to cell-cycle progression (E2F targets, G2-M checkpoint). The trial failed to meet the prespecified 6-month PFS and best overall response targets. We hypothesize that TKI efficacy may be reliant upon an interplay between kinase inhibition and the ACC immune microenvironment, whereas programs promoting cell-cycle progression may contribute to TKI resistance. These observations suggest that trials evaluating CDK4/6 inhibition plus a VEGFR TKI should be considered.

Prognostic Significance of CD11b-, CD8-, and CD163-Positive Tumor-Infiltrating Immune Cells in Distal Bile Duct Cancer.

Background: Distal bile duct cancer is an aggressive malignancy. Tumor-infiltrating immune cells (TIICs) in the tumor microenvironment are crucial for predicting prognosis in various cancers. In this study, we analyzed TIICs based on CD11b, CD163, and CD8 expression, and evaluated their association with clinicopathologic factors and prognosis in distal bile duct cancer. Methods: A total of 90 patients who underwent curative resection for distal bile duct cancer were enrolled. We analyzed CD11b+ tumor-infiltrating myeloid cells (TIMs), CD163+ tumor-infiltrating macrophages (TAMs), and CD8+ tumor-infiltrating lymphocytes (TILs) using immunohistochemistry and tissue microarrays. The correlation between TIICs and clinicopathologic characteristics was assessed. Results: Low levels of CD11b+ TIMs (p < 0.001) and high levels of CD8+ TILs (p = 0.003) were significantly associated with improved overall survival (OS). A combined low level of CD11b+ TIMs and high level of CD8+ TILs was identified as an independent favorable prognostic factor (hazard ratio, 0.159; confidence interval, 0.061-0.410; p < 0.001). Conclusions: CD11b+ TIMs play a crucial role in the tumor microenvironment and the prognosis of distal bile duct cancer. The combined analysis of CD11b+ TIMs and CD8+ TILs can predict survival in patients with distal bile duct cancer.

Examining the influence of tumor-infiltrating macrophages on breast cancer outcomes and identifying relevant genes for diagnostic purposes

ObjectiveThe purpose of this research was to investigate how different types of immune cells impact the outlook of individuals with breast cancer, as well as identify the essential genes associated with immune cell subtype enrichment.MethodsThe Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used to obtain global transcriptome sequencing data sets of breast tissue. The study utilized the CIBERSORT algorithm to determine the presence of 22 different types of immune cells in both breast cancer tissue and normal breast tissue.Immune cell infiltration content was utilized to conduct univariate COX analysis in order to identify risk factors linked to breast cancer prognosis.ResultsUnivariate COX analysis indicates that Macrophages M1 and B cells naive are beneficial factors for the outlook of individuals with breast cancer (P < 0.05), while Macrophages M2 and Monocytes are detrimental factors for the prognosis of breast cancer patients (P < 0.05). The high infiltration group of macrophage M2 had a poorer prognosis compared to the low infiltration group (P < 0.001); Conversely, the high infiltration group of macrophage M1 had a better prognosis than the low infiltration group (P = 0.002).ConclusionThe study provided an overview of immune cell infiltration in breast cancer tissues, identifying macrophage M1 and macrophage M2 as potential factors in breast cancer development and progression. Additionally, genes associated with macrophage phenotype were analyzed, offering insights into macrophage polarization mechanisms.

Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor DLBCL

AbstractThe pathogenesis of myelocytomatosis oncogene (MYC) and B-cell lymphoma 2 (BCL2) double-expressor diffuse large B-cell lymphoma (DE-DLBCL) remains unclear. To investigate how MYC and BCL2 contribute to tumor aggressiveness, we analyzed tumors from 14 patients each with DE-DLBCL and non–DE-DLBCL using whole transcriptome sequencing. Validation was performed using publicly available data sets, tumor tissues from 126 patients, DLBCL cell lines, and a syngeneic mouse lymphoma model. Our transcriptome analysis revealed significantly elevated messenger RNA levels of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in DE-DLBCLs when compared with non–DE-DLBCLs (adjusted P value < .05). Transcriptomic analysis of public data sets and immunohistochemistry corroborated these findings, indicating increased levels of M2 macrophages but a reduction in T-cell infiltration in DE-DLBCLs when compared with non–DE-DLBCLs (all P < .05). CCR2 expression was observed mainly in tumor-infiltrating macrophages and not in DLBCL cells. Increased expression of CCL2 and CCR2 was significantly associated with a poor prognosis in patients with DLBCL. In the in vitro analyses, MYChigh/BCL2high DLBCL cells showed higher CCL2 expression and secretion than MYClow/BCL2low cells. MYC and BCL2 increased CCL2 expression and secretion by upregulation of nuclear factor κB p65 in DLBCL cells, and CCL2 promoted M2 polarization of macrophages. In a mouse lymphoma model, CCL2 contributed to the immunosuppressive microenvironment and tumor growth of MYChigh/BCL2high tumors. We demonstrated that the increased CCL2/CCR2 axis confers aggressiveness to DE-DLBCL by increasing M2 polarization and can be a potential therapeutic target.

Abstract B043: Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration

Abstract Background: Identification of effective immunomodulatory strategies remains a major unmet need in pancreatic ductal adenocarcinoma (PDAC). Agonistic anti-CD40 antibody (aCD40) exhibits direct cytotoxicity on tumor cells and augments antigen presentation. Subasumstat(SUB), a sumoylation inhibitor, augments innate and adaptive immune responses by increasing interferon signaling. We hypothesized that SUB+aCD40 combination would promote an effective antitumor immune response in an aggressive orthotopic model of PDAC. Methods: 2000 KPC 46 cells were implanted in the pancreas of four groups of F1 hybrid (initial survival, CD8+ T-cell depletion, and clodronate liposome depletion) and nude mice, enrolled when tumors had reached 3-5mm. The vehicle (veh) group received 80ul SUB vehicle (q48 hrs) and 100 ug blank IgG (qwk); SUB only group received 15mg/kg q48h; aCD40 only group received 100ug qwk; SUB+aCD40 group received 15mg/kg SUB q48h and 100 ug aCD40 qwk. Single cell RNA sequencing (scRNASeq) was conducted on n=2 tumors from each group. Immunohistochemistry (IHC) was performed on veh and SUB+aCD40 tumors for macrophage infiltration using F4/80. Flow cytometry (FC) was employed for the presence of lymphoid and myeloid markers in veh, SUB+aCD40, and clodronate depleted SUB+aCD40 (SUB+aCD40+clod) groups. Results: Mice receiving SUB+aCD40 had a significantly improved median survival over those receiving monotherapy or vehicle (24.5d vs. SUB:15.5d, aCD40:20d, veh: 19d). scRNASeq revealed increased tumor infiltrating CD8+ T-cell and myeloid populations when comparing veh and monotherapy to SUB+aCD40. F4/80 IHC confirmed increased macrophage infiltration in SUB+aCD40 vs veh (p = 0.01). The survival advantage conferred by SUB+aCD40 was preserved in both CD8+ T-cell depleted mice (p=0.025) and nude mice (p=0.037), while clodronate eliminated the treatment survival advantage (p=0.39). FC showed increased tumor macrophage/monocyte infiltration in SUB+aCD40 vs veh (veh:11.9%, SUB+aCD40:22.5%, p=0.03) and reduction in SUB+aCD40+clod (12.6%, p=0.01). CD8+ T-cell CD69 expression decreased in the SUB+aCD40+clod group (SUB+aCD40:19%, SUB+aCD40+clod:6%, p&amp;lt; 0.01). CD8 T-cell infiltration (SUB+aCD40:4.95%, SUB+aCD40+clod:3.85%, p=0.27) and CD4/CD8 T-cell ratio (veh: 1.26, SUB+aCD40:0.52, SUB+aCD40+clod:0.69, p=0.09) were not affected by clodronate. Conclusion: SUB+aCD40 therapy confers a survival advantage in the KPC46 model of PDAC in a T-cell independent manner by increasing macrophage/monocyte infiltration. SUB+aCD40 promotes CD8+ T-cell infiltration and activation, but they do not contribute to survival advantage, possibly due to exhaustion. Future work will include addition of therapeutics to mitigate/reverse T-cell exhaustion to harness both innate and adaptive immunity in treating PDAC. Citation Format: Asimina Courelli, Kevin Li, James Lee, Herve Tiriac, Yuan Chen, Andrew Lowy. Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B043.

Altered epitopes enhance macrophage-mediated anti-tumour immunity to low-immunogenic tumour mutations.

Personalized neoantigen therapy has shown long-term and stable efficacy in specific patient populations. However, not all patients have sufficient levels of neoantigens for treatment. Although somatic mutations are commonly found in tumours, a significant portion of these mutations do not trigger an immune response. Patients with low mutation burdens continue to exhibit unresponsiveness to this treatment. We propose a design paradigm for neoantigen vaccines by utilizing the highly immunogenic unnatural amino acid p-nitrophenylalanine (pNO2Phe) for sequence alteration of somatic mutations that failed to generate neoepitopes. This enhances the immunogenicity of the mutations and transforms it into a suitable candidate for immunotherapy. The nitrated altered epitope vaccines designed according to this paradigm is capable of activating circulating CD8+ T cells and inducing immune cross-reactivity against autologous mutated epitopes in different MHC backgrounds (H-2Kb, H-2Kd, and human HLA-A02:01), leading to the elimination of tumour cells carrying the mutation. After immunization with the altered epitopes, tumour growth was significantly inhibited. It is noteworthy that nitrated epitopes induce tumour-infiltrating macrophages to differentiate into the M1 phenotype, surprisingly enhancing the MHC II molecule presenting pathway of macrophages. Nitrated epitope-treated macrophages have the potential to cross-activate CD4+ and CD8+ T cells, which may explain why pNO2Phe can enhance the immunogenicity of epitopes. Meanwhile, the immunosuppressive microenvironment of the tumour is altered due to the activation of macrophages. The nitrated neoantigen vaccine strategy enables the design of vaccines targeting non-immunogenic tumour mutations, expanding the pool of potential peptides for personalized and shared novel antigen therapy. This approach provides treatment opportunities for patients previously ineligible for new antigen vaccine therapy.

NAD+ Metabolism Reprogramming Mediates Irradiation-Induced Immunosuppressive Polarization of Macrophages

Purposeradiotherapy stands as an important complementary treatment for head and neck squamous cell carcinoma (HNSCC), yet it does not invariably result in complete tumor regression. The infiltration of immunosuppressive macrophages is believed to mediate the radiotherapy resistance, which mechanism remains largely unexplored. This study aimed to elucidate the role of immunosuppressive macrophages during radiotherapy and the associated underlying mechanisms. Materials and MethodsMale C3H mice bearing syngeneic SCC-VII tumor were received irradiation (2 × 8Gy). The impact of irradiation on tumor-infiltrating macrophages were assessed. Bone marrow derived macrophages were evaluated in differentiation, proliferation, migration, and inflammatory cytokines after treatment of irradiated tumor culture medium (irCM) and irradiated tumor derived extracellular vesicles (irTEVs). A comprehensive metabolomics profiling of the irTEVs was conducted using liquid chromatography-mass spectrometry, while key metabolites were investigated the mechanism in macrophage in vitro and in vivo. ResultsRadiotherapy on SCC-VII syngeneic graft tumors increased polarization of both M1 and M2 macrophages in tumor microenvironment and drove infiltrated macrophages towards an immunosuppressive phenotype. Irradiation-induced polarization and immunosuppression of macrophages were dependent on irTEVs which delivered an increased amount of nicotinamide (NAM) to macrophages. NAM directly bound to the NF-κB transcriptional activity regulator USP7, through which NAM reduced translocation of NF-κB into the nucleus, thereby decreasing the release of cytokines IL6 and IL8. Increased enzyme activity of nicotinamide phosphoribosyl transferase (NAMPT) which is the rate-limiting enzyme of NAD+ metabolism, contributed to the irradiation-induced accumulation levels of NAM in irradiated HNSCC and irTEVs. Inhibition of NAMPT decreased NAM levels in irTEVs and increased radiotherapy sensitivity through alleviating immunosuppressive function of macrophages. ConclusionsRadiotherapy could induce NAD+ metabolic reprogramming of HNSCC cells, which regulate macrophage towards an immunosuppressive phenotype. Pharmacological targeting NAD+ metabolism might be a promising strategy for radiotherapy sensitization of HNSCC.

The impact of CLDN18.2 expression on effector cells mediating antibody-dependent cellular cytotoxicity in gastric cancer

Activating antibody-dependent cellular cytotoxicity (ADCC) by targeting claudin-18 isoform 2 (CLDN18.2) using zolbetuximab, a monoclonal antibody against CLDN18.2, has been considered a promising novel therapeutic strategy for gastric cancer (GC). However, the impact of CLDN18.2 expression on natural killer (NK) cells and monocytes/macrophages—crucial effector cells of ADCC—in GC has not been fully investigated. In the present study, we assessed the impact of CLDN18.2 expression on clinical outcomes, molecular features, and the frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, in GC by analyzing our own GC cohorts. The expression of CLDN18.2 did not significantly impact clinical outcomes of GC patients, while it was significantly and positively associated with Epstein–Barr virus (EBV) status and PD-L1 expression. The frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, were comparable between CLDN18.2-positive and CLDN18.2-negative GCs. Importantly, both CLDN18.2 expression and the number of tumor-infiltrating NK cells were significantly higher in EBV-associated GC compared to other molecular subtypes. Our findings support the effectiveness of zolbetuximab in CLDN18.2-positive GC, and offer a novel insight into the treatment of this cancer type, highlighting its potential effectiveness for CLDN18.2-positive/EBV-associated GC.

High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment.

Enrichment of tumor-associated macrophages (TAMΦs) in the tumor microenvironment correlates with worse clinical outcomes in triple-negative breast cancer (TNBC) patients, prompting the development of therapies to inhibit TAMΦ infiltration. However, the lackluster efficacy of CCL2-based chemotaxis blockade in clinical trials suggests that a new understanding of monocyte/macrophage infiltration may be necessary. Here we demonstrate that random migration, and not only chemotaxis, drives macrophage tumor infiltration. We identified tumor- associated monocytes (TAMos) that display a dramatically enhanced migration capability, induced rapidly by the tumor microenvironment, that drives effective tumor infiltration, in contrast to low-motility differentiated macrophages. TAMo, not TAMΦ, promotes cancer cell proliferation through activation of the MAPK pathway. IL-6 secreted both by cancer cells and TAMo themselves enhances TAMo migration by increasing dendritic protrusion dynamics and myosin- based contractility via the JAK2/STAT3 signaling pathway. Independent from CCL2 mediated chemotaxis, IL-6 driven enhanced migration and pro-proliferative effect of TAMo were validated in a syngeneic TNBC mouse model. Depletion of IL-6 in cancer cells significantly attenuated monocyte infiltration and reversed TAMo-induced cancer cell proliferation. This work reveals the critical role random migration plays in monocyte driven TAMΦ enrichment in a tumor and pinpoints IL-6 as a potential therapeutic target in combination with CCL2 to ameliorate current strategies against TAMΦ infiltration.

FGFR2 fusion/rearrangement is associated with favorable prognosis and immunoactivation in patients with intrahepatic cholangiocarcinoma.

Increasing evidence highlights that fibroblast growth factor receptor 2 (FGFR2) fusion/rearrangement shows important therapeutic value for patients with intrahepatic cholangiocarcinoma (ICC). This study aims to explore the association of FGFR2 status with the prognosis and immune cell infiltration profiles of patients with ICC. A total of 226 ICC tissue samples from patients who received surgery at the Department of Liver Surgery at Zhongshan Hospital, Fudan University, were collected retrospectively and assigned to a primary cohort (n = 152) and validation cohort (n = 74) group. Fluorescence in situ hybridization was performed to determine FGFR2 status. Multiplex immunofluorescence (mIF) staining and immunohistochemistry were performed to identify immune cells. Thirty-two (14.2%) ICC tissues presented with FGFR2 fusion/rearrangement. FGFR2 fusion/rearrangement was associated with low levels of carcinoembryonic antigen (CEA, P = .026) and gamma glutamyl transferase (γ-GGT, P = .003), low TNM (P = .012), CNLC (P = .008) staging as well as low tumor cell differentiation (P = .016). Multivariate COX regression analyses revealed that FGFR2 fusion/rearrangement was an independent protective factor for both overall survival (OS) and relapse-free survival in patients with ICC. Furthermore, correlation analysis revealed that an FGFR2 fusion/rearrangement was associated with low levels of Tregs and N2 neutrophils and high levels of N1 neutrophils infiltrating into tumors but not with CD8+ T-cell or macrophage tumor infiltration. FGFR2 fusion/rearrangement may exert a profound impact on the prognosis of ICC patients and reprogram the tumor microenvironment to be an immune-activated state. FGFR2 status may be used for ICC prognostic stratification and as an immunotherapeutic target in patients with ICC.