Here, we show that combined use of the EZH2 inhibitor GSK126 and the CDK4/6 inhibitor abemaciclib synergistically enhances antitumoral effects in preclinical GBM models. Dual blockade led to HIF1α upregulation and CalR translocation, accompanied by massive impairment of mitochondrial function. Basal oxygen consumption rate, ATP synthesis, and maximal mitochondrial respiration decreased, confirming disrupted endoplasmic reticulum-mitochondrial homeostasis. This was paralleled by mitochondrial depolarization and upregulation of the UPR sensors PERK, ATF6α, and IRE1α. Notably, dual EZH2/CDK4/6 blockade also reduced 3D-spheroid invasion, partially inhibited tumor growth in ovo, and led to impaired viability of patient-derived organoids. Mechanistically, this was due to transcriptional changes in genes involved in mitotic aberrations/spindle assembly (Rb, PLK1, RRM2, PRC1, CENPF, TPX2), histone modification (HIST1H1B, HIST1H3G), DNA damage/replication stress events (TOP2A, ATF4), immuno-oncology (DEPDC1), EMT-counterregulation (PCDH1) and a shift in the stemness profile towards a more differentiated state. We propose a dual EZH2/CDK4/6 blockade for further investigation.

Background: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. Methods: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2^del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. Results: Compared to wildtype mice, Rag2^del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2⁻MHC-II^lo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. Conclusions: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.

Adipose tissue includes various cell types beyond the typical adipocytes. The stromal vascular fraction (SVF) contains mesenchymal stem cells (MSCs), pericytes, and endothelial cells, which can be isolated from adipose tissue by mechanical and enzymatic methods. The composition of the SVF is heterogeneous, and donor factors such as sex, age, body mass index (BMI), and harvesting site are associated with variations in cellular composition and viability. The expression of specific surface markers, which determine the immunophenotype of the cells, can also vary. In this study, we investigated the effects of donor age, BMI, and harvesting site on cell yield, viability, and size. Our results showed that BMI significantly influenced cell yield and size, with overweight and obese donors yielding more cells than normal-weight donors. Additionally, cells isolated from the adipose tissue of the thighs/legs were larger than those from other areas. Flow cytometry showed considerable variability in SVF composition among donors. These results emphasize that SVF donor characteristics have a significant impact on cell yield, viability, and cell size, with the immunophenotype being highly donor-dependent. Understanding these factors is crucial for optimizing cell yield and defining populations for therapeutic applications of SVF cells.

FDA-approved immune checkpoint inhibitors (ICIs) are the state-of-the-art treatment for mismatch-repair deficient tumors (dMMR). Their immunomodulatory effects in a preventive setting, are poorly studied. In this study, we used two ICIs against PD-L1 or LAG-3 in a preclinical mouse model of dMMR-driven carcinogenesis. Msh2^{loxP/loxP; TgTg(Vil1-cre} mice without clinical signs of tumor development (i.e. age <12 weeks) received repeated applications (n = 8, 28-day interval) of anti-PD-L1, anti-LAG-3 (2.5 mg/kg bw, i.p.) or isotype (anti-IgG1, 2.5 mg/kg bw, i.p.). Blood phenotyping, tumor microenvironment, and hematopoiesis, and presence of procoagulant extracellular vesicles (EV) were studied. Prophylactic ICI application significantly prolonged overall survival (OS) of Msh2^{loxP/loxP; TgTg(Vil1-cre} mice (OS: anti-PD-L1: 54.4 wks and anti-LAG-3: 58.4 wks vs. ctrl 35.8 wks). Circulating exhausted and regulatory T cells were significantly lower in the ICI groups. Splenic exhaustion markers showed correlating results. Outgrowing tumors showed an inflammatory response-related gene signature. Accompanying immunofluorescence confirmed data and identified reduced numbers of tumor-infiltrating regulatory granulocytes in late-onset tumors. Alterations in bone marrow hematopoiesis accompanied this massive immune modulation, indicating successful prevention of myeloid-shifted hematopoiesis. Plasma coagulation of EVs was not significantly altered in the ICI groups. Preventive ICI prolongs overall survival of cancer-prone mice. The sustained immune modulation and normal bone marrow hematopoiesis may pre-sensitize late-onset dMMR tumors to a second round of immunotherapy. Hence, we highlight the importance of preventive strategies for germline MMR mutation carriers and recommend improved screening of patients eligible for prophylactic ICIs to improve long-term outcomes.

While the lung is considered an efficient site for stopping the larvae of the acute Schistosoma spp. infection phase from migrating through extensive inflammatory responses in the surrounding tissues, little is known about these processes. To date, the highest resistance to infection has been achieved in experimental studies with radiation-attenuated cercariae immunization, which elicits a strong Th1/Th2 response in the lung and results in up to 80% protection. Based on our own studies demonstrating a systemic, unpolarized Th1/Th2 response resulting from infection with male or female Schistosoma mansoni, we hypothesize that this atypical immune response is already detectable during the pulmonary passage of parasite larvae. Therefore, we examined the immune milieu in the lungs of mice caused by migrating schistosome larvae, either male or female (single-sex groups) or male + female (bisexual control), 4 and 16 days after infection in bronchoalveolar lavage and lung tissue by flow cytometry, qPCR, and multiplex analyzes. Our results show only minor differences in the inflammatory profile between the single-sex groups but significant differences compared with the bisexual control group. Both single-sex infected groups have increased expression of inflammatory markers in lung tissue, higher numbers of cytotoxic T cells (day 4 post-infection) and more T helper cells (day 16 post-infection), compared with the bisexual control group. A single-sex infection, regardless of whether it is an infection with male or female cercariae, causes an immune milieu in the lung that is clearly different from an infection with both sexes. In terms of identifying therapeutic targets to achieve resistance to re-infection, it is of great scientific interest to identify the differences in the inflammatory potential of male or female and male + female parasites.

Background: The CDK4/6 inhibitor abemaciclib is an FDA-approved agent and induces T-cell-mediated immunity. Previously, we confirmed the therapeutic potential of abemaciclib on mismatch repair-deficient (dMMR) tumors in mice. Here, we applied a prophylactic administration/dosage setting using two preclinical mouse models of dMMR-driven cancer. Methods: Mlh1- /- and Msh2loxP/loxP mice received repeated prophylactic applications of abemaciclib mesylate (75 mg/kg bw, per oral) as monotherapy or were left untreated. Blood phenotyping and multiplex cytokine measurements were performed regularly. The tumor microenvironment was evaluated by immunofluorescence and Nanostring-based gene expression profiling. Numbers, size and immune composition and activity of extracellular vesicles (EVs) were studied at the endpoint. Findings: Prophylactic abemaciclib-administration delayed tumor development and significantly prolonged overall survival in both mouse strains (Mlh1- /- : 50.0 wks vs. control: 33.9 wks; Msh2loxP/loxP;TgTg(Vil1-cre: 58.4 wks vs. control 44.4 wks). In Mlh1- /- mice, pro-inflammatory cytokines (IL-2, IL-6) significantly increased, whereas IL-10 and IL-17A decreased. Circulating and splenic exhausted and regulatory T cell numbers were significantly lower in the abemaciclib groups. Deeper analysis of late-onset tumors revealed activation of the Hedgehog and Notch signaling in Mlh1- /- mice, and activation of the MAPK pathway in Msh2loxP/loxP;TgTg(Vil1-cre mice. Still, arising tumors had fewer infiltrating myeloid-derived suppressor cells (vs. control). Notably, prophylactic abemaciclib-administration prevented secretion of procoagulant EVs but triggered release of immunomodulatory EVs in Mlh1- /- mice. Interpretation: Prophylactic abemaciclib prolongs survival via global immunomodulation. Prophylactic use of abemaciclib should be considered further for individuals with inherited dMMR. Funding: This work was supported by grants from the German research foundation [DFG grant number: MA5799/ 2-2] and the Brigitte und Dr. Konstanze Wegener-Stiftung to CM.