Preparation of a Single-Cell Suspension from Tumor Biopsy Samples for Single-Cell RNA Sequencing.

An essential requirement for single-cell RNA sequencing in cancer is the preparation of high-quality single-cell suspensions from the tumor tissue. In this work, various methods of dissociation of tumor biopsy specimens were analyzed and developed to obtain a cell suspension with at least 80% viability. It was found that the optimal conditions for sample preparation are mechanical dissociation followed by incubation with a collagenase/hyaluronidase mixture with addition of DNAase I for 60 min. Thus, we optimize the approach for preparing single-cell suspensions from the tumor biopsy tissue for single-cell RNA sequencing.

[Radioimmunotherapy: current state of the problem].

This review presents modern data about one of the promising direction in treatment of malignancies, which is called radioimmunotherapy. The special attention is paid to search of new targets and development of radiopharmaceuticals that will allow improving quality and accuracy of the directed impact on tumors of various localizations.

EpCAM-CD24+ circulating cells associated with poor prognosis in breast cancer patients.

Following the discovery of circulating tumor cells (CTCs) in the peripheral blood of cancer patients, CTCs were initially postulated to hold promise as a valuable prognostic tool through liquid biopsy. However, a decade and a half of accumulated data have revealed significant complexities in the investigation of CTCs. A challenging aspect lies in the reduced expression or complete loss of key epithelial markers during the epithelial-mesenchymal transition (EMT). This likely hampers the identification of a pathogenetically significant subset of CTCs. Nevertheless, there is a growing body of evidence regarding the prognostic value of such molecules as CD24 expressing in the primary breast tumor. Herewith, the exact relevance of CD24 expression on CTCs remains unclear. We used two epithelial markers (EpCAM and cytokeratin 7/8) to assess the count of CTCs in 57 breast cancer patients, both with (M0mts) and without metastasis (M0) during the follow-up period, as well as in M1 breast cancer patients. However, the investigation of these epithelial markers proved ineffective in identifying cell population expressing different combinations of EpCAM and cytokeratin 7/8 with prognostic significance for breast cancer metastases. Surprisingly, we found CD24+ circulating cells (CCs) in peripheral blood of breast cancer patients which have no epithelial markers (EpCAM and cytokeratin 7/8) but was strongly associated with distant metastasis. Namely, the count of CD45-EpCAM-CK7/8-CD24+ N-cadherin-CCs was elevated in both groups of patients, those with existing metastasis and those who developed metastases during the follow-up period. Simultaneously, an elevation in these cell counts beyond the established threshold of 218.3 cells per 1 mL of blood in patients prior to any treatment predicted a 12-fold risk of metastases, along with a threefold decrease in distant metastasis-free survival over a 90-month follow-up period. The origin of CD45-EpCAM-CK7/8-CD24+ N-cadherin-CCs remains unclear. In our opinion their existence can be explained by two most probable hypotheses. These cells could exhibit a terminal EMT phenotype, or it might be immature cells originating from the bone marrow. Nonetheless, if this hypothesis holds true, it's worth noting that the mentioned CCs do not align with any of the recognized stages of monocyte or neutrophil maturation, primarily due to the presence of CD45 expression in the myeloid cells. The results suggest the presence in the peripheral blood of patients with metastasis (both during the follow-up period and prior to inclusion in the study) of a cell population with a currently unspecified origin, possibly arising from both myeloid and tumor sources, as confirmed by the presence of aneuploidy.

Evaluation of HER2/neu Expression in Metastatic Axillary Lymph Node Tissue of Breast Cancer Patients Using [99mTc]Tc-(HE)3-G3.

Anatomic visualization and molecular typing of metastatic regional lymph nodes in breast cancer patients are a serious clinical challenge in modern oncology. According to the results of previous studies, [99mTc]Tc-(HE)3-G3 has proven to be a promising diagnostic agent in differentiating the HER2/neu receptor status in primary breast tumors (p < 0.05, Mann-Whitney test). In this regard, the purpose of this study is to explore the possibilities of using [99mTc]Tc-(HE)3-G3 to determine the HER2/neu receptor status in the metastatic axillary lymph nodes (mALNs) of breast cancer patients. The study was conducted using clinical material from 20 breast cancer patients (T2-4N1-3M0-1) before systemic therapy (10 patients with positive and 10 patients with negative HER2/neu expression in mALNs) who underwent SPECT/CT scan 4 h after the administration of [99mTc]Tc-(HE)3-G3. Morphological and immunohistochemical studies of mALNs with assessment of the HER2/neu status were performed on all patients. We found that mALN-to-background and mALN-to-latissimus dorsi muscle ratios for [99mTc]Tc-(HE)3-G3 uptake 4 h after its administration may be used for typing of the HER2/neu status in mALNs of breast cancer patients (p < 0.05, Mann-Whitney test). In that case, sensitivity and specificity for the mALN-to-background ratio were identical at 80%, with the threshold value being > 12.25.

The Evolution of Targeted Radionuclide Diagnosis of HER2-Positive Breast Cancer.

This review examines the evolution of the radionuclide diagnosis of HER2-positive breast cancer using various compounds as a targeting module in clinical practice: from full-length antibodies to a new group of small synthetic proteins called alternative scaffold proteins. This topic is of especial relevance today in view of the problems attendant to the detection of breast cancer with HER2/neu overexpression, which, in most cases, introduce errors in the treatment of patients. The results of clinical studies of radiopharmaceuticals based on affibody molecules, ADAPTs, and DARPins for SPECT and PET have demonstrated good tolerability of the compounds, their rapid excretion from the body, and the possibility to differentiate tumor sites depending on the HER2/neu status. This indicates that targeted radionuclide diagnosis holds promise and the need to continue research in this direction.

Molecular subtype conversion in CTCs as indicator of treatment adequacy associated with metastasis-free survival in breast cancer.

Molecular subtype of breast cancer has a great clinical significance and used as one of the major criteria for therapeutic strategy. Recently, for anticancer therapy, the trend for oncologists is the predominant determination of biomarkers in the existing foci of the disease. In the case of adjuvant therapy prescribed for distant metastases prevention, CTCs could be a suitable object for investigation. CTCs as one of the factors responsible for tumor metastatic potential could be more convenient and informative for evaluation of hormone receptors, Ki-67 and HER2 expression, which are determine molecular subtype in breast cancer patient. In our study, we aimed to investigate the molecular subtype discordance between the primary tumor and CTCs in breast cancer patients. We established conversion of molecular subtype in most of the cases. Namely, conversion was detected in 90% of untreated patients and in 82% of breast cancer patients treated by neoadjuvant chemotherapy. At the same time, molecular subtype conversions in patients treated by neoadjuvant chemotherapy were more diverse. Molecular subtype conversions resulted more often in the unfavorable variants in circulating tumor cells. We stratified all patients according to the adequacy of treatment against converted CTCs molecular subtype. Our study revealed that good response to neoadjuvant chemotherapy observed in case of adequate therapy, namely, when chemotherapy scheme was sufficient against CTCs. It turned out that patients with inadequate therapy were characterized by decreased simulated 5-year metastasis-free survival compared to patients who received appropriate therapy. Thus, detection of molecular subtype conversion in circulating tumor cells could be a perspective tool for optimization of antitumor therapy.

[Radionuclide teranostic of malignancies].

This review shows some basic information regarding the relatively new and one of the promising areas, called "theranostics' and also discusses its basic principles. Special attention is paid to the study of approaches in selecting the most appropriate in the application of radioisotopes for solutions appropriate diagnostic and therapeutic applications. Also presented the main directions in the work with monoclonal antibodies and their use in radionuclide theranostics.