Unexplained infertility (UI) continues to pose a diagnostic challenge, affecting a considerable proportion of reproductive-aged women. Increasing evidence suggests that oxidative stress (OS) may contribute to impaired female reproductive function. Malondialdehyde (MDA) is a lipid peroxidation marker, while total antioxidant capacity (T-AOC) reflects overall antioxidant defense. Evaluating these biomarkers may help to better understand the role of OS in UI and the potential benefit of antioxidant therapy. A prospective observational study included 30 women diagnosed with primary unexplained infertility. Serum levels of MDA and T-AOC were measured at baseline and after a period of antioxidant supplementation lasting 1-7 months (duration mode: 3 months). All participants received standardized antioxidant therapy consisting of vitamin E (50 mg/day), zinc (15 mg/day), coenzyme Q10 (15 mg/day), and selenium (70 µg/day). Participants with known causes of infertility were excluded. Nonparametric statistical tests were used to evaluate changes in oxidative stress markers before and after supplementation and to compare subgroups with and without comorbidities. Median baseline MDA concentration was 228.2 ng/mL and decreased significantly after antioxidant supplementation to 173.9 ng/mL (p < 0.001), with a reduction observed in 90% of participants. Median T-AOC increased slightly from 23.9 U/mL to 26.2 U/mL, but the change was not statistically significant (p = 0.735). No significant differences in oxidative stress markers were found between women with and without comorbidities, although higher baseline MDA levels were observed in participants with endometriosis (stage I-II). A significant decrease in MDA after supplementation was seen both in women with endometriosis (p = 0.005) and without it (p < 0.001). Women with unexplained infertility demonstrate biochemical evidence of oxidative stress, reflected by elevated MDA levels. Antioxidant supplementation was associated with a significant reduction in lipid peroxidation, suggesting a potential therapeutic role of antioxidants in UI. Combined assessment of MDA and T-AOC may provide useful insight into oxidative imbalance in infertility, although larger controlled studies are needed.

Abstract Tumor heterogeneity limits the reproducibility and interpretability of in vitro ovarian cancer models. In this study, we established a comparative framework that intentionally modulates stromal-induced complexity through two complementary approaches: scaffold-free stromal co-culture with patient-derived cancer-associated fibroblasts (CAFs) or fibroblast cell line WS1, and scaffold-based micro-scaffolds printed using two-photon polymerization (2PP) with poly(ethylene glycol) diacrylate (PEGDA). We analyzed four ovarian cancer cell lines (A2780, SKOV3, COV362 and OV7,) stemness/epithelial-mesenchymal transition (EMT), and TGF-β. We then evaluated their 3D spheroid formation and growth dynamics across different seeding densities. In ultra-low-attachment plates, SKOV3, COV362, and OV7 formed round, compact spheroids across different seeding densities, while A2780 produced loose aggregates. Co-culturing with early-passage CAFs or WS1 fibroblast cell line (in 2:1, 1:1, and 1:2 ratios of cancer cells : fibroblast) rescued A2780‘s ability to form spheroids and increased the compactness of OV7 and COV362 in a line- and ratio-dependent manner in co-culture with CAFs. Compared to 2D cultures, 3D spheroids exhibited higher expression of stemness/EMT and angiogenesis related genes. In contrast, the PEGDA scaffolds (with pore sizes of 65, 100, and 130 µm) standardized early attachment and surface coverage for both spheroid-competent line SKOV3, and a line that does not typically form spheroids, A2780. These scaffolds also shifted EMT/stemness gene expression without elevating vascular endothelial growth factor (VEGF) levels compared to scaffold-free 3D cultures. Together, these results provide practical guidance: utilize fibroblast co-culture to study the stromal-induced complexity and cell-to-cell interactions, including the rescue of cell lines that typically do not form spheroids, and employ printed micro-scaffolds to control 3D culture variability.

Background/Objectives: Electrochemotherapy (ECT) is a reliable and potent technique for managing primary tumors; however, significant efforts are being made to characterize and improve the systemic immune response, which is crucial for metastasis prevention. Current evidence suggests that the advancement of ECT will depend on its integration with complementary immunomodulatory methods. Methods: In this study, we examined the combined effects of calcium-based electrochemotherapy (CaECT, 1.3 kV/cm × 100 µs, eight pulses delivered at 1 Hz repetition frequency) with dendritic cell vaccination (DCV). Lewis lung carcinoma (LLC1) was used as a tumor model. We characterized the effects of CaECT alone and in combination with DCV therapy on tumor growth, analyzed the changes in immune cell subpopulations, and studied the humoral immune response dynamics on day 10, 20, and 30. Given the limited effect of DCV, additional experiments were conducted with the chemotherapeutic drug cyclophosphamide (CP), known for its immunomodulatory properties. Results: Although CaECT demonstrated potent antitumor activity and induced a significant immune response, its combination with DCV did not result in enhanced therapeutic efficacy. The combination of CP also failed to improve median survival. Conclusions: It is concluded that CaECT is a promising alternative to standard ECT involving bleomycin or cisplatin. However, further optimization is necessary to enhance the therapeutic synergy of CaECT when combined with DCV.

Different patterns of immune and stromal interactions within the tumor microenvironment significantly impact tumor behavior and response to treatment. Recent research has identified distinct immune subtypes—immune desert, excluded, and inflamed—each associated with specific mechanisms that impede anti-tumor immune responses. In the era of precision medicine, accurately stratifying patients into these subtypes is essential for optimizing cancer management and personalizing immunotherapy. The research team at National Cancer Institute in Lithuania addresses critical gaps in this field by focusing on several key areas. Our first focus is on developing reliable and accessible tools for determining patient immune subtypes. We are integrating transcriptomics and histology data to create a robust subtyping pipeline, which we are validating with real-world data and streamlining for clinical use. Another aspect of our work involves creating translatable preclinical models for cancer immunotherapy through utilizing 3D cell cultures and biocompatible scaffolds. We are developing druggable syngeneic murine tumor models that accurately reflect the three immune subtypes, enhancing the relevance of preclinical studies. Using both pharmaceutical and non-pharmaceutical approaches, we are tailoring immunomodulatory interventions to address the unique characteristics of each immune subtype. Joint efforts in these research areas could generate translatable insights, contributing to more precise cancer therapies and overcoming resistance mechanisms.

BACKGROUND:Preclinical studies demonstrate that exercise reduces tumor incidence and growth. Rapid release of extracellular vesicles (EVs) during exercise suggests their potential role as mediators of exercise-induced systemic effects and physiological adaptation. This study investigated the impact of exercise-induced plasma EVs on tumor growth and immune tumor microenvironment in murine models of triple-negative breast cancer (TNBC): EO771 (a C57BL/6-derived TNBC cell line) and 4T1 (a BALB/c-derived TNBC cell line). METHODS:Size exclusion chromatography was used to isolate exercise-induced EVs from plasma of healthy female mice (BALB/c and C56BL/6, n = 30 per strain) that underwent ten 30-min moderate-intensity treadmill running sessions over 2 weeks. Nanoparticle tracking analysis, Western blot, and electron microscopy confirmed the presence of EVs in the samples. Tumor-bearing mice (n = 72 per strain) were administered with exercise-induced EVs before or/and after tumor implantation. Local and systemic immune responses were assessed using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and quantitative polymerase chain reaction (qPCR). RESULTS:Administration of exercise-induced EVs, particularly before tumor implantation, significantly suppressed tumor growth and reduced tumor burden in both TNBC models. In EO771, endpoint tumor volumes were 278-330 mm³ in treated groups compared to 799 mm³ in untreated (p < 0.0001), while in 4T1, treated groups showed volumes of 287-564 mm³ vs. 696 mm³ in untreated (p = 0.0002). Notable differences in tumor-infiltrating lymphoid and myeloid cell subpopulations indicated immunomodulatory effects of exercise-induced EVs, particularly in the 4T1 model, where their continuous administration significantly increased intratumoral cluster of differentiation 8 (CD8) T lymphocyte proportion (5.77% vs. 0.90% in untreated, p < 0.0001). Similarly, in the EO771 model, exercise-induced EVs administered before tumor implantation led to a marked rise in intratumoral CD8 T lymphocytes (2.24% vs. 1.08% in untreated, p = 0.0181). CONCLUSION:Our findings indicate that exercise-induced EV treatment elicits a pro-inflammatory antitumor immune response, suggesting a shift of immunologically cold TNBC tumors towards a more inflamed phenotype associated with better outcomes. Our study supports the further investigation of EVs as modulators of antitumor immunity and their potential utility in enhancing the efficacy of immunotherapy.

Background:Soluble PD-1 (sPD-1) and PD-L1 (sPD-L1) are emerging biomarkers in prostate cancer (PCa), yet their cellular origins and interactions within the tumor microenvironment (TME), particularly with NK cells, immunosuppressive Tregs, and MDSCs, remain poorly understood. This study also explores biomarker combinations for better clinically significant PCa stratification. Methods:A total of 88 PCa patients (pT2 and pT3) were enrolled. Plasma levels of sPD-1 and sPD-L1 were measured using ELISA. A control group of 41 healthy individuals was also included. Immune cell populations were characterized by flow cytometry. All parameters were evaluated preoperatively and three months post-radical prostatectomy (RP). Clinically significant PCa was defined as cases with International Society of Urological Pathology (ISUP) ≥ 3. Written informed consent was obtained from all participants. Results:sPD-1 exhibited a weak positive correlation with NK cells (r=0.27, p=0.03) in whole cohort, suggesting NK cells as a source of sPD-1. Higher initial sPD-L1 levels were significantly associated with shorter progression-free survival (PFS) (p<0.05), additionally in the biochemical recurrence (BCR) group, sPD-L1 showed a positive correlation with Tregs (r=0.73, p<0.05), highlighting their coordinated role in cancer progression. Postoperative changes in sPD-L1 divided patients into two distinct groups: one showed a decrease (p<0.001), indicating tumor-derived origins, while the other exhibited an increase, suggesting an alternative source (p<0.05). In patients with a postoperative decrease in sPD-L1, no correlation was found with pretreatment Tregs and MDSC subsets. However, strong associations between total G-MDSCs and M-MDSCs (r=-0.51, p=0.002) and between G-MDSC% and M-MDSC% (r=0.49, p=0.001) were observed, indicating a dynamic interplay between these myeloid subsets, which could contribute to immunosuppressive mechanisms regulating sPD-L1 levels. Moreover, a negative correlation between sPD-L1 and M-MDSC% was found in the BCR group (r=-0.72, p<0.05). The combination of preopreative levels of sPD-L1 with the G-MDSC fraction (best result: AUC=0.93, specificity 100%) demonstrated superior results for clinically significant prostate cancer stratification compared to the M-MDSC fraction. Combinations with Tregs showed poor predictive value. Conclusions:Our findings indicate that sPD-1 is primarily derived from NK cells, while sPD-L1 levels are modulated by Tregs and interactions between MDSC subsets. These insights contribute to a deeper understanding of immune evasion in PCa and may facilitate the development of biomarker-driven approaches for disease monitoring and targeted therapies.

Introduction: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. Methods: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. Results: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. Conclusions: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.

Unexplained infertility (UI) remains a diagnostic challenge affecting a significant proportion of women of reproductive age. Vascular endothelial growth factor (VEGF), a key mediator of angiogenesis and inflammation, has been implicated in reproductive and immune processes. This prospective observational study evaluated serum VEGF levels and allergen sensitization (via ALEX2 macroarray) in 70 women—51 with UI and 19 fertile controls—to assess VEGF’s potential as a biomarker in UI. Median VEGF concentrations were higher in women with UI compared to fertile controls (128.6 pg/mL vs. 82.5 pg/mL), though not statistically significantly. However, sensitized women showed significantly elevated VEGF levels compared to non-sensitized peers (115.9 pg/mL vs. 85.7 pg/mL, p = 0.028), and a stepwise increase in VEGF was observed with rising allergy severity (p = 0.045). Sensitization to pet allergens, particularly cat allergen Fel d 1, was associated with the highest VEGF levels. A literature review confirmed wide variability in VEGF concentrations and the lack of standardized norms. While VEGF alone may not serve as a definitive biomarker for infertility, elevated levels may reflect an underlying inflammatory state. Our findings suggest VEGF testing could support broader clinical evaluation in women with UI, especially in the presence of allergic sensitization.

The diagnostic and prognostic potential of histopathology can be significantly enhanced with three-dimensional (3D) imaging. Histopathology relies on the gold-standard 2D inspection of hematoxylin and eosin (H&E)-stained tissue sections with a brightfield microscope. We introduce a novel methodology termed nonlinear-H&E (N-H&E) that utilizes inherent optical sectioning of nonlinear microscopy for 3D volumetric investigations. Multimodal multiphoton excitation fluorescence (MPF) and third-harmonic generation (THG) microscopy is employed to image fluorescent and nonfluorescent structures. The optical sections are used to create 3D brightfield-equivalent H&E volumes by employing a generative adversarial network (GAN)-based image-to-image translation. N-H&E generates realistic 2D and 3D brightfield images with enhanced axial resolution. Furthermore, polarimetric second-harmonic generation (P-SHG) is applied to visualize the 3D collagen architecture. The overlay of volumetric P-SHG and GAN-generated H&E images provides an enriched view of tumor microenvironment for 3D histopathology. Reconstructed images meet standards for visual quality, structural accuracy, and clinical assessment reliability.

This study aimed to evaluate the diagnostic potential of soluble Programmed Death Ligand 1 (sPD-L1) and Programmed Death 1 (sPD-1) molecules in plasma, along with urinary mRNA biomarkers—Prostate-Specific Membrane Antigen (PSMA), Prostate Cancer Antigen 3 (PCA3), and androgen receptor (AR) genes—for identifying clinically significant prostate cancer (PCa), defined as pathological stage 3. In a cohort of 68 PCa patients, sPD-L1 and sPD-1 levels were quantified using ELISA, while mRNA transcripts were measured by RT-qPCR. Results highlight the potential of integrating these liquid-based biomarkers. In particular, the combination of sPD-L1, sPD-1, and AR demonstrated the most significant improvement in diagnostic performance, increasing the area under the curve (AUC) from 0.65 to 0.81 and sensitivity from 60% to 88%, compared to AR alone. PSMA demonstrated an AUC of 0.82 and a specificity of 52.8%, which improved to an AUC of 0.85 and a specificity of 94.4% with the inclusion of sPD-L1 and sPD-1. Similarly, PCA3 achieved an AUC of 0.75 and a specificity of 53.8%, increasing to an AUC of 0.78 and a specificity of 76.9% when combined with these biomarkers. Incorporating sPD-L1 into a three-gene panel further elevated the AUC from 0.74 to 0.94. These findings underscore the value of multimodal liquid-based diagnostic panels in improving the management of clinically significant PCa.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy and unique long-term outcomes in treating relapsed/refractory hematological malignancies in both pediatric and adult patients. However, a subset of patients remains unresponsive, with disease progression continuing despite treatment. Moreover, CAR-T therapy has shown limited success in treating solid tumors. Accumulating evidence on the role of various lymphocyte subpopulations in antitumor immunity suggests that cytokine-induced killer (CIKs) cells represent a promising alternative for adoptive cell therapy. A key advantage of CIKs is their low toxicity and the presence of unrestricted MHC cytotoxicity against both hematological and solid malignancies. In our study, we hypothesize that modifying CIKs with CARs would enhance their antitumor efficacy compared to unmodified CIKs. Using a unique retroviral vector containing the CD34 or CD14 marker gene, we successfully generated CD19 CAR-expressing lymphocyte populations, including both classically activated T lymphocytes and CIKs. We then compared their functional properties by analyzing lymphocyte subpopulations based on markers such as CD3, CD4, CD8, CD56, CD62L, and CD45RA, as well as their ability to specifically recognize CD19-positive targets. Additionally, we assessed cytokine production, including IFN-γ (via ELISA and intracellular staining), IL-2, TNF-α, and IL-10 (via intracellular staining). Our findings indicate that CIKs can be efficiently modified using a retroviral vector while maintaining functional activity. CD19 CAR-CIK populations contained higher numbers of NKT (CD3⁺CD56⁺) and CD3⁺CD8⁺ lymphocytes compared to CD19 CAR-T cells. However, the proportion of naïve-like T cells (CD62L⁺CD45RA⁺) and effector memory RA⁺ T cells (CD62L⁻CD45RA⁺) remained similar in both CAR-modified populations. Additionally, CD19 CAR-CIKs exhibited a slightly increased ability to produce IFN-γ and TNF-α in response to CD19⁺ tumor cells. Despite that, key questions remain regarding the functionality of CD19 CAR-CIKs in an immunosuppressive tumor microenvironment, particularly in the presence of high levels of TGF-β, IL-10, and PD-L1, which may inhibit their activity or lead to exhaustion. Our future research will focus on overcoming these challenges to enhance the therapeutic potential of CAR-CIKs.

Continuous passaging of cancer cell lines can drive phenotypic and genotypic divergence, potentially compromising the reliability of such models. In this study, we show that two late-passage strains (S1 and S2) of ovarian cancer cell line SKOV3, although authenticated via short tandem repeat (STR) profiling as identical, exhibit substantial differences in morphology, transcriptomic signatures, ability to form 3D cultures and chemotherapeutic responses. Notably, S1 formed compact 3D spheroids and exhibited enhanced epithelial-mesenchymal transition (EMT) pathway activity, whereas S2 displayed a more proliferative, MYC-driven phenotype with larger spheroid structures requiring higher seeding densities. Transcriptomic analysis revealed pathways associated with hypoxia, EMT and angiogenesis in 3D culture, highlighting the complexity introduced by dimensionality in tumour modelling. Critically, S1 showed higher sensitivity to doxorubicin than S2 (IC50 of 0.12 µM versus 1.28 µM, P=0.0001), indicating how clonal evolution can confound drug-response assays. Ultimately, our findings suggest that although STR profiling remains essential for cell line authentication, functionally distinct subpopulations can arise and coexist within the same culture, and their isolation may reveal divergent phenotypes that compromise reproducibility in preclinical cancer research.

Colorectal cancer (CRC) is a heterogeneous disease characterized by a diverse tumor microenvironment comprising immune and non-immune components. Acquired mutations and phenotypic plasticity in the tumor microenvironment create a diversity of cells whose interactions contribute to tumor progression and create resistance to anticancer therapy through various activated mechanisms. At the molecular level, four subtypes of colorectal cancer (CMS1-4) have been validated, differing in their aggressive characteristics and immune infiltration. With the rapid development of oncology science and the development of new cancer treatment strategies, more attention is given to the microenvironment surrounding cancer cells. Since the tumor is densely infiltrated by host immune cells, especially macrophages, it could be a promising target for developing new treatment strategies. This study aims to explore how inhibition of CRC cell stemness properties with specific inhibitors could modulate macrophage polarization, shifting the immune balance from protumoral M2 type to the antitumoral M1 type. We evaluated the effect of four stemness inhibitors - salinomycin, SB-431542, JIB-04, and napabucasin - on CMS4 CRC cell lines (HCT116 and SW620) and human peripheral blood-derived macrophages in an indirect co-culture model. Our results demonstrate that CMS4 CRC cell lines induced distinct macrophage polarization patterns, with HCT116 promoting M2 macrophages and SW620 leaning toward M1 profile. The combination of stemness inhibitors reduced stemness markers (CD133, CD44) in colorectal cancer cells and shifted macrophage polarization toward an M1 phenotype, particularly in co-culture with HCT116. These findings suggest that stemness inhibitors not only target cancer cells but also modulate the immune microenvironment by reprogramming macrophages.

Introduction: Epidemiological evidence highlights the role of physical activity in breast cancer is crucial due to its potential to reduce cancer risk and improve outcomes. However, there is still a need for a more comprehensive understanding of how lifestyle factors, such as exercise, can modulate the anticancer immune response. Aim: Given the relationship between host physical status and immune fitness, this preclinical study aimed to explore the impact of exercise-induced extracellular vesicles (EVs) on the immune tumor microenvironment and breast cancer progression. Material and Methods: Mice underwent a treadmill-based exercise regimen, with plasma collected post-exercise for EVs isolation. Two transplantable breast cancer models (4T1 and E0771) were treated with exercise-induced EVs prophylactically and therapeutically, along with monitoring tumor growth and profiling of systemic and local immune microenvironment. Results: Notably, regular exercise led to a significant increase in plasma EVs levels in active mice. Treatment with exercise-induced extracellular vesicles resulted in a marked delay in tumor growth (approximately 30%, p<0.05) compared to untreated controls. Analysis of lymphoid and myeloid cell subpopulations revealed discernible immunomodulatory effects of EVs on the tumor microenvironment. Further examination of tumor-infiltrating lymphocytes suggested that the observed delay in tumor growth in the treatment group may be attributed to the influx of cytotoxic CD8+ T cells. Conclusions: This study unveils impact of physical activity on EVs production and their potential therapeutic role in breast cancer. Our findings suggest that extracellular vesicles could serve as an immunomodulatory treatment to trigger inflammation in the tumor bed.

The spatial distribution of tumor infiltrating lymphocytes (TILs) defines several histologically and clinically distinct immune subtypes—desert (no TILs), excluded (TILs in stroma), and inflamed (TILs in tumor parenchyma). To date, robust classification of immune subtypes still requires deeper experimental evidence across various cancer types. Here, we aimed to investigate, define, and validate the immune subtypes in melanoma by coupling transcriptional and histological assessments of the lymphocyte distribution in tumor parenchyma and stroma. We used the transcriptomic data from The Cancer Genome Atlas melanoma dataset to screen for the desert, excluded, and inflamed immune subtypes. We defined subtype-specific genes and used them to construct a subtype assignment algorithm. We validated the two-step algorithm in the qPCR data of real-world melanoma tumors with histologically defined immune subtypes. The accuracy of a classifier encompassing expression data of seven genes (immune response-related: CD2, CD53, IRF1, and CD8B; and stroma-related: COL5A2, TNFAIP6, and INHBA) in a validation cohort reached 79%. Our findings suggest that melanoma tumors can be classified into transcriptionally and histologically distinct desert, excluded, and inflamed subtypes. Gene expression-based algorithms can assist physicians and pathologists as biomarkers in the rapid assessment of a tumor immune microenvironment while serving as a tool for clinical decision making.

Objectives: To assess the role of soluble PD-L1 and PD-1 in combination with mRNA transcripts of PCA3, PSMA, AR genes in diagnostics of clinically significant prostate cancer (PCa). Methods: For 68 PCa patients, plasma sPD-L1 and sPD-1 were measured by ELISA method and the urinary PSMA, PCA3 and AR were tested using RT-qPCR. Results: PSMA and AR were iden-tified as the most reliable biomarkers for predicting clinically significant prostate cancer, with AUCs of 0.81 and 0.78, respectively. Additionally, the expression levels of PSMA and PCA3 were associated with the pT3 stage. Despite the diagnostic potential of mRNA transcripts alone, the ad-dition of sPD-L1 and sPD-1 significantly enhanced diagnostic accuracy. Combination doubled the specificity of PCA3 from 44.1% to 88.0%, compared to PCA3 alone. sPD-L1, sPD-1 and AR resulted in the best distinction between clinically significant and insignificant prostate cancer, achieving an AUC of 0.97, an accuracy of 0.95, a sensitivity of 100%, a specificity of 95%, revealing the potential of AR in distinguishing clinically significant PCa, more effectively than PSMA and PCA3. Our find-ings highlight the significant potential of sPD-1 in enhancing diagnostic accuracy when added to a triple gene panel, achieving an AUC of 0.90 compared to an AUC of 0.78 without sPD-1. This un-derscores the value of sPD-1, which has been less explored than sPD-L1 in cancer diagnostics. Con-clusions: sPD-L1 and sPD-1 has a potential to significantly enhance the accuracy of PCa diagnostics when added to the diagnostic panel, alongside PSMA, PCA3, and AR mRNA transcripts.

Dendritic cell (DC) based immunotherapy is one of the strategies to combat cancer invoking a patient's immune system. This form of anticancer immunotherapy employs adjuvants to enhance the immune response, triggering mechanisms of innate immunity and thus increase immunotherapeutic efficiency. A conventional adjuvant for DCs maturation during production of anticancer vaccines is bacterial LPS. Nevertheless, synthetic dsRNAs were also shown to stimulate different receptors on innate immune cells and to activate immune responses through induction of cytokines via toll-like receptors. In our study we investigated the potential of Larifan as dsRNA of natural origin to stimulate maturation of DCs with proinflammatory (possible antitumoral) activity and to compare these immunostimulatory properties between Larifan’s fractions with different molecular lengths. To explore the suitability of this product for therapy, it is necessary to study the properties of its different fractions and compare them to standard adjuvants. We investigated the effect of Larifan’s fractions on immune system stimulation in vivo by monitoring the survival time of tumor-bearing mice. Murine DCs produced in vitro using Larifan and its fractions together with tumor antigens during production were also characterized. All Larifan fractions resulted in inducing high expression of immunogenic markers CD40, CD80, CD86, CCR7, MHC II and lower secretion of the immunosuppressive cytokine IL-10, compared to the maturation with LPS in mDCs. The lowest expression of tolerogenic gene Ido1 and highest expression of the immunogenic genes Clec7a, Tnf, Icosl, Il12rb2, Cd209a were characteristic to the unfractionated dsRNA and short fraction FR15. In the mouse model the best overall survival rate was observed in mice treated with medium-length FR9 and FR15. We can state that both Larifan and its fractions were superior to LPS as vaccine adjuvants in stimulating phenotype and functional activity of mature DCs. DCs maturation using these factors induces a valuable anticancer immune response.

PurposeThe aim of this study was to assess the role of sPD-L1 and sPD-1 as potential biomarkers in prostate cancer (PCa). The association of the values of these soluble proteins were correlated to the clinical data: stage of disease, Gleason score, biochemical recurrence etc. For a comprehensive study, the relationship between sPD-L1 and sPD-1 and circulating immune cells was further investigated.Methods A total of 88 patients with pT2 and pT3 PCa diagnosis and 41 heathy men were enrolled. Soluble sPD-L1 and sPD-1 levels were measured in plasma by ELISA method. Immunophenotyping was performed by flow cytometry analysis.Results Our study’s findings demonstrate that PCa patients had higher levels of circulating sPD-L1 and sPD-1 comparing to healthy controls (p < 0.001). We found a statistically significant (p < 0.05) relationship between improved progression free survival and lower initial sPD-L1 values. Furthermore, patients with a lower sPD-1/sPD-L1 ratio were associated with a higher probability of disease progression (p < 0.05). Additionally, a significant (p < 0.05) association was discovered between higher Gleason scores and elevated preoperative sPD-L1 levels and between sPD-1 and advanced stage of disease (p < 0.05). A strong correlation (p < 0.05), between immunosuppressive CD4+CD25+FoxP3+ regulatory T cells and baseline sPD-L1 was observed in patients with unfavorable postoperative course of the disease, supporting the idea that these elements influence each other in cancer progression. In addition to the postoperative drop in circulating PD-L1, the inverse relationship (p < 0.05), between the percentage of M-MDSC and sPD-L1 in patients with BCR suggests that M-MDSC is not a source of sPD-L1 in PCa patients.Conclusion Our findings suggest the potential of sPD-L1 as a promising prognostic marker in prostate cancer.