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.

Innate lymphoid cells (ILCs), the complements of diverse CD4 T helper cells, help maintain tissue homeostasis by providing a link between innate and adaptive immune responses. While pioneering studies over the last decade have advanced our understanding how ILCs influence adaptive immune responses to pathogens, far less is known about whether the adaptive immune response feeds back into an ILC response. In this study, we isolated ILCs from blood of healthy donors, fine-tuned culture conditions, and then directly challenged them with human adenoviruses (HAdVs), with HAdVs and host defense proteins (HDPs) or neutralizing antibodies (NAbs), to mimic interactions in a host with pre-existing immunity. Additionally, we developed an ex vivo approach to identify how bystander ILCs respond to the uptake of HAdVs ± neutralizing antibodies by monocyte-derived dendritic cells. We show that ILCs take up HAdVs, which induces phenotypic maturation and cytokine secretion. Moreover, NAbs and HDPs complexes modified the cytokine profile generated by ILCs, consistent with a feedback loop for host antiviral responses and potential to impact adenovirus-based vaccine efficacy.
Copyright © 2022 Paris, Mennechet and Kremer.

The purpose of this study was to investigate allogenic immune responses following the transplantation of insulin-producing cells (IPCs) differentiated from human adipose tissue-derived stem cells (hAT-MSCs) into humanized mice.
hAT-MSCs were isolated from liposuction aspirates obtained from HLA-A2-negative healthy donors. These cells were expanded and differentiated into IPCs. HLA-A2-positive humanized mice (NOG-EXL) were divided into 4 groups: diabetic mice transplanted with IPCs, diabetic but nontransplanted mice, nondiabetic mice transplanted with IPCs and normal untreated mice. Three million differentiated cells were transplanted under the renal capsule. Animals were followed-up to determine their weight, glucose levels (2-h postprandial), and human and mouse insulin levels. The mice were euthanized 6-8 weeks posttransplant. The kidneys were explanted for immunohistochemical studies. Blood, spleen and bone marrow samples were obtained to determine the proportion of immune cell subsets (CD4+, CD8+, CD16+, CD19+ and CD69+), and the expression levels of HLA-ABC and HLA-DR.
Following STZ induction, blood glucose levels increased sharply and were then normalized within 2 weeks after cell transplantation. In these animals, human insulin levels were measurable while mouse insulin levels were negligible throughout the observation period. Immunostaining of cell-bearing kidneys revealed sparse CD45+ cells. Immunolabeling and flow cytometry of blood, bone marrow and splenic samples obtained from the 3 groups of animals did not reveal a significant difference in the proportions of immune cell subsets or in the expression levels of HLA-ABC and HLA-DR.
Transplantation of IPCs derived from allogenic hAT-MSCs into humanized mice was followed by a muted allogenic immune response that did not interfere with the functionality of the engrafted cells. Our findings suggest that such allogenic cells could offer an opportunity for cell therapy for insulin-dependent diabetes without immunosuppression, encapsulation or gene manipulations.
© 2022. The Author(s).

Innate lymphoid cells (ILCs), the complements of diverse CD4 T helper cells, help maintain tissue homeostasis by providing a link between innate and adaptive immune responses. While pioneering studies over the last decade have advanced our understanding how ILCs influence adaptive immune responses to pathogens, far less is known about whether the adaptive immune response feeds back into an ILC response. In this study, we isolated ILCs from blood of healthy donors, fine-tuned culture conditions, and then directly challenged them with human adenoviruses (HAdVs), with HAdVs and host defence proteins (HDPs) or neutralizing antibodies (NAbs), to mimic interactions in a host with pre-existing immunity. Additionally, we developed an ex vivo approach to identify how bystander ILCs respond to the uptake of HAdVs ± neutralizing antibodies by monocyte-derived dendritic cells. We show that ILCs take up HAdVs, which induces phenotypic maturation and cytokine secretion. Moreover, NAbs and HDPs complexes modified the cytokine profile generated by ILCs, consistent with a feedback loop for host antiviral responses and potential to impact adenovirus-based vaccine efficacy. h4>Author Summary/h4> Several studies have shown the importance of innate lymphoid cells (ILCs) both from an immune and physiological point of view, in particular for their role in the maintenance of tissue integrity, pathogens clearance, or in the establishment of immune tolerance. Our study focuses on the role of ILCs during direct challenge with prototype vaccines based on human adenoviruses (HAdVs) ± host defence proteins (HDPs) or neutralizing antibodies (NAbs) to mimic interactions in a host with pre-existing immunity. In parallel, through an ex vivo approach we observe how bystander ILCs respond to the uptake of HAdVs ± NAbs by monocyte-derived dendritic cells. We show that ILCs take up HAdVs, which induces pro- inflammatory and antiviral responses through phenotypic maturation and cytokine secretion. Moreover, HAdV-NAb and HAdV-HDP complexes modified the cytokine profile generated by ILCs, consistent with a feedback loop for host antiviral responses and potential to impact HAdV vaccine efficacy.

Recently, a paradigm shift has been established for oncolytic viruses (OVs) as it was shown that the immune system plays an important role in the specific killing of tumor cells by OVs. OVs have the intrinsic capacity to provide the right signals to trigger anti-tumor immune responses, on the one hand by delivering virus-derived innate signals and on the other hand by inducing immunogenic cell death (ICD), which is accompanied by the release of various damage-associated molecules from infected tumor cells. Here, we determined the ICD-inducing capacity of Talimogene laherparepvec (T-VEC), a herpes simplex virus type 1 based OV, and benchmarked this to other previously described ICD (e.g., doxorubicin) and non-ICD inducing agents (cisplatin). Furthermore, we studied the capability of T-VEC to induce the maturation of human BDCA-1+ myeloid dendritic cells (myDCs). We found that T-VEC treatment exerts direct and indirect anti-tumor effects as it induces tumor cell death that coincides with the release of hallmark mediators of ICD, while simultaneously contributing to the maturation of BDCA-1+ myDCs. These results unequivocally cement OVs in the category of cancer immunotherapy.