Aberrant overexpression of PIWI/piRNA pathway proteins is shown for many types of tumors. Interestingly, these proteins are downregulated in testicular germ cell tumors (TGCTs) compared to normal testis tissues. Here, we used germline and TGCT markers to assess the piRNA biogenesis and function in TGCTs and their precursor germ cell neoplasia in situ (GCNIS). We used small RNA deep sequencing, qRT-PCR, and mining public RNAseq/small RNA-seq datasets to examine PIWI/piRNA gene expression and piRNA biogenesis at four stages of TGCT development: (i) germ cells in healthy testis tissues, (ii) germ cells in testis tissues adjacent to TGCTs, (iii) GCNIS cells and (iv) TGCT cells. To this end, we studied three types of samples: (a) healthy testis, (b) testis tissues adjacent to two types of TGCTs (seminomas and nonseminomas) and containing both germ cells and GCNIS cells, as well as (c) matching TGCT samples. Based on our analyses of small RNA-seq data as well as the presence/absence of expression correlation between PIWI/piRNA pathway genes and germline or TGCT markers, we can suggest that piRNA biogenesis is intact in germ cells present in healthy adult testes, and adjacent to TGCTs. Conversely, GCNIS and TGCT cells were found to lack PIWI/piRNA pathway gene expression and germline-like piRNA biogenesis. However, using an in vitro cell line model, we revealed a possible role for a short PIWIL2/HILI isoform expressed in TGCTs in posttranscriptional regulation of the youngest members of LINE and SINE classes of transposable elements. Importantly, this regulation is also implemented without involvement of germline-like biogenesis of piRNAs. Though further studies are warranted, these findings suggest that the conventional germline-like PIWI/piRNA pathway is lost in transition from germ cells to GCNIS cells.

Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the formation of a chemotherapeutic agent inside cancer cells. However, despite great efforts, promising preliminary results, and a long period of time (over 25 years) since the first mention of this method, GDEPT has not yet reached the clinic. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. The advent of checkpoint immunotherapy has yielded new highly promising avenues of study in cancer therapy. For such therapy, it seems reasonable to use combinations of different immunomodulators alongside traditional methods, such as chemotherapy and radiotherapy, as well as GDEPT. In this review, we focused on non-viral gene immunotherapy systems combining the intratumoral production of toxins diffused by GDEPT and immunomodulatory molecules. Special attention was paid to the applications and mechanisms of action of the granulocyte-macrophage colony-stimulating factor (GM–CSF), a cytokine that is widely used but shows contradictory effects. Another method to enhance the formation of stable immune responses in a tumor, the use of danger signals, is also discussed. The process of dying from GDEPT cancer cells initiates danger signaling by releasing damage-associated molecular patterns (DAMPs) that exert immature dendritic cells by increasing antigen uptake, maturation, and antigen presentation to cytotoxic T-lymphocytes. We hypothesized that the combined action of this danger signal and GM–CSF issued from the same dying cancer cell within a limited space would focus on a limited pool of immature dendritic cells, thus acting synergistically and enhancing their maturation and cytotoxic T-lymphocyte attraction potential. We also discuss the problem of enhancing the cancer specificity of the combined GDEPT–GM–CSF–danger signal system by means of artificial cancer specific promoters or a modified delivery system.

It is established that BCG vaccination results in the development of both a specific immune response to mycobacterial infections and a nonspecific (heterologous) immune response, designated as trained immunity (TRIM), to other pathogens. We hypothesized that local BCG immunization may induce an early immune response in bone marrow and spleen innate immunity cells. The early transcriptomic response of various populations of innate immune cells, including monocytes, neutrophils, and natural killer (NK) cells, to BCG vaccination was examined. To this end, C57Bl/6J mice were subcutaneously immunized with 10⁶ CFU of BCG. Three days following BCG administration, the three cell populations were collected from the control and BCG-vaccinated groups using FACS. All cell populations obtained were utilized for the preparation and sequencing of RNA-seq libraries. The analysis of FACS data revealed an increase in the proportion of splenic NK cells and monocytes 3 days post-vaccination. Transcriptomic analysis revealed the deregulation of genes associated with the regulation of immune response (according to Gene Ontology terms) in NK cells, monocytes, and unsorted bone marrow cells. Two NK cell-specific immune ligands (Tnfsf14 and S100a8) and two bone marrow-specific immune receptors (C5ar1 and Csf2rb) were identified among differentially expressed genes. No alterations were identified in neutrophils in either their percentage or at the transcriptomic level. Thus, in this study, we demonstrated that BCG vaccination provides an early increase in the proportion of murine bone marrow and spleen immune cell populations, as well as transcriptomic alterations in monocytes, NK cells, and non-sorted bone marrow cells. This early innate immune response may be beneficial for enhancing TRIM.

The search for effective anti-cancer therapies is one of the most significant goals of modern medicine. The combination of oncolytic viruses (OV) and mRNA immunoadjuvants can significantly improve the outcome or even substitute traditional immunotherapy. In addition to the direct OV-mediated cytotoxic elimination of tumor cells, both OV and mRNA immunoadjuvants can significantly alter the immunosuppressive tumor microenvironment (TME) supporting cancer cells and unleash the immune response against the malignant cells. The present study is aimed at assessing the therapeutic effects of recombinant vesicular stomatitis virus (rVSV) and lipid nanoparticles (LNP) delivering mRNA coding for murine interleukin-12 (mIL12) and granulocyte-macrophage colony-stimulating factor (mGMCSF) (mRNA-LNP) in colorectal carcinoma CT26-induced tumors both as independent therapies and in combination with each other. The results of the in vivo experiment on BALB/c mice demonstrated that rVSV monotherapy did not have a significant effect, with the tumor growth inhibition index (TGII) ranging from 13.7 to 29.8% on days 6–10 after the therapy start. While monotherapy with mRNA-LNP was more effective (TGII of 48.6–53.7%), it was the therapy combining the two approaches (rVSV and mRNA-LNP) that resulted in the highest TGII of 66.7% on day 10. While these results can be further improved by optimizing the experimental design, they show the great potential of combination immunotherapy for the treatment of oncological diseases.

It is established that BCG vaccination results in the development of both a specific immune response to mycobacterial infections and a nonspecific (heterologous) immune response, designated as trained immunity (TRIM), to other pathogens. We hypothesized that local BCG immunization may induce early immune response of bone marrow and spleen innate immunity cells. The early transcriptomic response of various populations of innate immune cells, including monocytes, neutrophils, and natural killer (NK) cells, to BCG vaccination was examined. To this end, C57Bl/6J mice were subcutaneously immunized with 10⁶ CFU of BCG. Three days following BCG administration, the three cell populations were collected from the control and BCG-vaccinated groups by FACS. All cell populations obtained were utilized for the preparation and sequencing of RNA-seq libraries. The analysis of FACS data revealed an increase in the proportion of splenic NK cells and monocytes 3 days post-vaccination. Transcriptomic analysis revealed deregulation of genes associated with regulation of immune response (according to Gene Ontology terms) in NK cells, monocytes, and unsorted bone marrow. Two NK cell-specific immune ligands (Tnfsf14 and S100a8) and two bone marrow-specific immune receptors (C5ar1 and Csf2rb) were identified among differentially expressed genes. No alterations were identified in neutrophils in either the percentage or at the transcriptomic level. Thus, in this study we demonstrated that BCG vaccination provides the early increase in the proportion of murine bone marrow and spleen immune cell populations, as well as the transcriptomic alterations in monocytes, NK cells, and non-sorted bone marrow. This early innate immune response may be beneficial for enhancing TRIM.

Background/Objectives: Large language models (LLMs) have been proposed as a means of converting unstructured electronic medical records (EMRs) into structured datasets. However, concerns regarding the reliability of these models in non-English clinical text and their capacity to generate novel insights remain unresolved. We aimed to utilize an LLM to identify a hypothetical “Luminal B poor-prognosis” breast cancer subgroup (LPP) based on progesterone receptor (PR), the Ki-67 proliferation index, and grade characteristics, while concurrently validating the LLM’s accuracy. Methods: We retrospectively compiled the EMRs on 7756 female breast cancer patients from five Moscow oncology centers. An LLM with a domain-engineered prompt extracted eight clinicopathological variables (Ki-67, estrogen receptor (ER)/PR Allred status, HER2 status, grade, relapse dates, and multiple primaries). The accuracy of the model was validated in 366 randomly sampled cases against oncologist annotations using Intraclass Correlation Coefficient (ICC) and weighted κ. Following data post-processing, the complete-case cohort (n = 2347) and the HR+/HER2− stage I–III sub-cohort (n = 1419) were analyzed. Survival was estimated with Kaplan–Meier/log-rank and modeled with Cox regression (adjusted for age, stage, and treatment). Ki-67 was modeled continuously; prespecified LPP definitions were compared. Results: LLM–human agreement was high (Ki-67 ICC = 0.882; grade κ = 0.887; ER κ = 0.997; PR κ = 0.975; HER2 κ = 0.935). Date extraction was characterized by a high degree of missing data. In HR+/HER2− stage I–III disease, ER < 5 was non-prognostic; however, PR < 4 and Ki-67 ≥ 40% were indicative of inferior survival (HR 2.25 and 1.85). The most effective LPP definition (PR < 4 and Ki-67 ≥ 40%) identified a subgroup (~5.3%) of patients with markedly poorer outcomes (age, stage, and treatment adjusted HR 2.60, 95% CI 1.53–4.43) compared to the Luminal B (HER2−) subgroup. Conclusions: The developed LLM has demonstrated the ability to reliably structure non-English EMRs and enable discovery of clinically meaningful subgroups. The discovered LPP phenotype defines a small, high-risk subset warranting external validation. Given the retrospective, single-system design of the study, it is imperative to interpret the discovered phenotype features as hypothesis-generating, rather than as definitive evidence.