The tumor microenvironment plays a pivotal role in cancer progression and therapeutic resistance, with tumor-associated macrophages significantly influencing immune suppression and tumor growth. Colorectal cancers (CRC) classified as Consensus Molecular Subtype 4 (CMS4) and triple-negative breast cancers subsets are particularly characterized by a mesenchymal phenotype, immune exclusion, and extensive macrophage infiltration. This study aimed to investigate how targeting cancer cell stemness with specific inhibitors could modulate macrophage polarization in CRC in vitro and breast cancer in vivo, potentially shifting the immune balance from pro-tumor M2-like to anti-tumor M1-like macrophages.
We used four stemness inhibitors-salinomycin, SB-431542, JIB-04, and napabucasin-each targeting different pathways (Wnt/β-catenin, TGF-β, histone demethylation, and STAT3, respectively), to evaluate their effects on CMS4 CRC cell lines (HCT116 and SW620) and human peripheral blood-derived macrophages in an indirect co-culture model.
Our results showed that CMS4 CRC cell lines induced distinct macrophage polarization patterns, with HCT116 promoting M2-like macrophages and SW620 leaning toward M1-like profile. Notably, the combination of stemness inhibitors reduced stemness markers (CD133, CD44) in colorectal cancer cells and shifted macrophage polarization toward an M1-like phenotype, particularly in co-culture with HCT116. In vivo studies using the syngeneic immunocompetent EO771 breast cancer mouse model demonstrated that combination of stemness inhibitors increased the M1/M2 macrophage ratio.
Our study highlights the dual potential of stemness inhibitors to target both cancer cells and the immune microenvironment. These findings offer promising strategies for enhancing favorable immunomodulation in mesenchymal-like colorectal tumors.
Copyright © 2025 Butkute, Baltramonaitis, Malmige, Darinskas, Pasukoniene and Mlynska.

Pentraxin 3 (PTX3), a long pentraxin and a humoral pattern recognition molecule (PRM), has been demonstrated to be protective against Aspergillus fumigatus, an airborne human fungal pathogen. We explored its mode of interaction with A. fumigatus, and the resulting implications in the host immune response. Here, we demonstrate that PTX3 interacts with A. fumigatus in a morphotype-dependent manner: (a) it recognizes germinating conidia through galactosaminogalactan, a surface exposed cell wall polysaccharide of A. fumigatus, (b) in dormant conidia, surface proteins serve as weak PTX3 ligands, and (c) surfactant protein D (SP-D) and the complement proteins C1q and C3b, the other humoral PRMs, enhance the interaction of PTX3 with dormant conidia. SP-D, C3b or C1q opsonized conidia stimulated human primary immune cells to release pro-inflammatory cytokines and chemokines. However, subsequent binding of PTX3 to SP-D, C1q or C3b opsonized conidia significantly decreased the production of pro-inflammatory cytokines/chemokines. PTX3 opsonized germinating conidia also significantly lowered the production of pro-inflammatory cytokines/chemokines while increasing IL-10 (an anti-inflammatory cytokine) released by immune cells when compared to the unopsonized counterpart. Overall, our study demonstrates that PTX3 recognizes A. fumigatus either directly or by interplaying with other humoral PRMs, thereby restraining detrimental inflammation. Moreover, PTX3 levels were significantly higher in the serum of patients with invasive pulmonary aspergillosis (IPA) and COVID-19-associated pulmonary aspergillosis (CAPA), supporting previous observations in IPA patients, and suggesting that it could be a potential panel-biomarker for these pathological conditions caused by A. fumigatus.
© 2024. The Author(s).

Recent studies have pointed to the role of Krüpple-like factor 12 (KLF12) in cancer-associated processes, including cancer proliferation, apoptosis, and metastasis. However, the role of KLF12 in tumor immunity remains obscure. Here, we found that KLF12 expression was significantly higher in non-small cell lung cancer (NSCLC) cells with higher programmed death-ligand 1 (PD-L1) expression. Additionally, a positive correlation between KLF12 and PD-L1 was observed in clinical patient tumor tissues. By chromatin immunoprecipitation (ChIP) analysis, KLF12 was identified to bind to the CACCC motif of the PD-L1 promoter. Overexpression of KLF12 promoted PD-L1 transcription, whereas silencing of KLF12 inhibited PD-L1 transcription. Furthermore, signal transducer and activator of transcription 1 (STAT1)- and STAT3-triggered PD-L1 transcription was abolished in the absence of KLF12, and KLF12 knockdown weakened the binding of STAT1 and STAT3 to the PD-L1 promoter. Mechanistically, KLF12 physically interacted with P300, a histone acetyltransferase. In addition, KLF12 silencing reduced P300 binding to the PD-L1 promoter, which subsequently caused decreased acetylation of histone H3. PD-L1 transcription driven by KLF12 overexpression was eliminated by EP300 silencing. In immunocompetent mice, KLF12 knockout inhibited tumor growth and promoted infiltration of CD8+ T cells. However, this phenomenon was not observed in immunodeficient mice. Overall, this study reveals KLF12-mediated transcriptional regulation of PD-L1 in NSCLC; targeting KLF12 may be a potential therapeutic strategy for NSCLC.
© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Macrophages play a pivotal role in tumor immunity. We report that reprogramming of macrophages to tumor-associated macrophages (TAMs) promotes the secretion of exosomes. Mechanistically, increased exosome secretion is driven by MADD, which is phosphorylated by Akt upon TAM induction and activates Rab27a. TAM exosomes carry high levels of programmed death-ligand 1 (PD-L1) and potently suppress the proliferation and function of CD8+ T cells. Analysis of patient melanoma tissues indicates that TAM exosomes contribute significantly to CD8+ T cell suppression. Single-cell RNA sequencing analysis showed that exosome-related genes are highly expressed in macrophages in melanoma; TAM-specific RAB27A expression inversely correlates with CD8+ T cell infiltration. In a murine melanoma model, lipid nanoparticle delivery of small interfering RNAs (siRNAs) targeting macrophage RAB27A led to better T cell activation and sensitized tumors to anti-programmed cell death protein 1 (PD-1) treatment. Our study demonstrates tumors use TAM exosomes to combat CD8 T cells and suggests targeting TAM exosomes as a potential strategy to improve immunotherapies.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Combination therapies that aim to improve the clinical efficacy to immune checkpoint inhibitors have led to the need for non-invasive and early pharmacodynamic biomarkers. Positron emission tomography (PET) is a promising non-invasive approach to monitoring target dynamics, and programmed death-ligand 1 (PD-L1) expression is a central component in cancer immunotherapy strategies. [18F]DK222, a peptide-based PD-L1 imaging agent, was investigated in this study using humanized mouse models to explore the relationship between PD-L1 expression and therapy-induced changes in cancer.
Cell lines and xenografts derived from three non-small cell lung cancers (NSCLCs) and three urothelial carcinomas (UCs) were used to validate the specificity of [18F]DK222 for PD-L1. PET was used to quantify anti-programmed cell death protein-1 (PD-1) therapy-induced changes in PD-L1 expression in tumors with and without microsatellite instability (MSI) in humanized mice. Furthermore, [18F]DK222-PET was used to validate PD-L1 pharmacodynamics in the context of monotherapy and combination immunotherapy in humanized mice bearing A375 melanoma xenografts. PET measures of PD-L1 expression were used to establish a relationship between pathological and immunological changes. Lastly, spatial distribution analysis of [18F]DK222-PET was developed to assess the effects of different immunotherapy regimens on tumor heterogeneity.
[18F]DK222-PET and biodistribution studies in mice with NSCLC and UC xenografts revealed high but variable tumor uptake at 60 min that correlated with PD-L1 expression. In MSI tumors treated with anti-PD-1, [18F]DK222 uptake was higher than in control tumors. Moreover, [18F]DK222 uptake was higher in A375 tumors treated with combination therapy compared with monotherapy, and negatively correlated with final tumor volumes. In addition, a higher number of PD-L1+ cells and higher CD8+-to-CD4+ cell ratio was observed with combination therapy compared with monotherapy, and positively correlated with PET. Furthermore, spatial distribution analysis showed higher [18F]DK222 uptake towards the core of the tumors in combination therapy, indicating a more robust and distinct pattern of immune cell infiltration.
[18F]DK222-PET has potential as a non-invasive tool for monitoring the effects of immunotherapy on tumors. It was able to detect variable PD-L1 expression in tumors of different cancer types and quantify therapy-induced changes in tumors. Moreover, [18F]DK222-PET was able to differentiate the impact of different therapies on tumors.
© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.