The investigation of in-vitro response of cell cultures derived from tumor material of individual patients with similar tumor localizations to photodynamic treatment is presented. Tumor types included in the research were renal cell carcinoma, melanoma and alveolar, synovial, lypo- and osteo- sarcomas. Long-term observations of treatment-induced morphological changes in cells were performed by means of digital holographic microscopy. A substantial variance in response of cells of individual patients with similar tumor types and localizations to photodynamic treatment with the same dose has been observed. These peculiarities are indicative of the demand to personalized protocols of photodynamic treatment. The elevated resistance of cells of some patients to treatment at high doses highlights potential limitations of photodynamic therapy for some patients. Digital holographic microscopy is shown to be an informative label-free noninvasive tool allowing for long-term monitoring of cell samples in vitro and providing quantitative information on necrosis rate and loss of cellular dry mass. The developed methodology can be generalized for analysis of cellular response to various therapies.

Tumorigenesis is related to the generation of heterogeneous tumor cell population, which is the result of genetic and epigenetic alterations followed by clonal selections and subsequent expansion. In basic studies genetic, histological and morphological diversity of different clones within a patient's neoplasm and specifics of their interrelation with patient's immune system are investigated mostly on the models of tumors of epithelial origin. Mesenchymal tumors such as soft tissue and bone-derived sarcomas (STBS) have been poorly studied in this regard. The molecular genetic methods used to examine intratumoral heterogeneity do not currently provide insight into which portion of the identified subclones are able to grow autonomously. Limiting dilution cloning demonstrates the existence of self-regulating tumor cells in the population and can serve as an independent prognostic predictor of poor prognosis. Intratumoral heterogeneity results not only in differences in growth dynamics, gene expression, and phenotypic markers, but also in the resistance to treatment, especially immunotherapy, thus causing tumor eluding immune escape. The changes that accompany this process can be affected by the cellular immune system, resulting in an imbalance between populations. The variations in the population composition of immune system cells are now widely debated as a predictor of response to immunotherapy, which is of obvious interest for sarcomas, where the effectiveness of chemotherapy is low and the prognosis is unfavorable, especially in case of metastatic disease development. The search for new predictive markers of disease prognosis and treatment efficacy is an important task, to which this study is focused. Our results demonstrate that clonogenic tumor characteristics such as clonogenic potential is independent predictor of unfavorable prognosis in cases of cancer and correlate with the clinical characteristics of the tumor such as overall survival (OS) and progression free survival (PFS). It was found that patients with clonogenic sarcomas had a lower content of activated cytotoxic T-lymphocytes (CTL) with the CD3(+)CD8(+)HLA-DR+ phenotype and an increased number of natural NK killers (p < 0.05) compared to nonclonogenic tumors. In addition, according to our data, a high neutrophil to lymphocyte ratio (NLR), a low value of major T-lymphocyte populations, and a higher number of natural killer cells (NK) in the blood can be negative prognostic factors for the immunotherapy of this disease.

The malignancy progression is an evolutionary process in which tumor clones are selected and competed for the duration of the disease. Intratumor heterogeneity is one of the key problems in the development of treatment methods for cancer patients. In this study we obtained metastatic soft tissue and bone sarcomas (STBSs) cultures from 54 patients, performed in vitro cloning and randomly selected 83 clones. Cloning was successful in 22 cases (40.7%). STBSs cultures with a high clonogenic potential (CP) were characterized by greater proliferative activity and increased Aldehyde dehydrogenase (ALDH) expression. We studied the transcription activity of the following cancer-testis genes (CTG): MAGE, NY-ESO-1, PRAME, GAGE, SSX1, HAGE1, PASD1, SCP1, SEMG1, SLLP1 and SPANXA1. The SEMG1 expression wasn't registered in any studied case. CTG activity wasn't observed in 10 cases out of 52 (19,2%) STBS cultures. We observed CTG activation and increased transcription activity in 82 STBSs clones. Clustering by the gene profile has revealed three different patterns: 1 st - with low expression CTG, 2nd - with co-expression GAGE1, PASD1 and PRAME, 3d - with co-expression SLLP1 and GAGE1. The last two clusters included most cloned cell lines and their clones. CP of STBSs cell lines was associated with the parameters of patients overall survival (OS) at comparable progression-free survival (PFS). Among patients with STBSs with the high CP, median OS was 7.6 months (min 0.7 - max 11.0 months). In the group with the low CP, OS did not reach the median value by the end of the five-year observation period. PFS was 5.6 months (min 0.2 - max 19.2 months) in the first group and 3.2 months (min 0.3- max 71.3 months) in the second group. Resistance to therapeutic doses of chemotherapy drugs was correlated with CP cultures STBSs. We suggest that chemotherapy-resistant clones are pre-existing in the tumor rather than being formed under the influence of chemotherapy. Highly aggressive metastatic sarcomas may be a promising candidate for immunotherapy against cancer-testis antigens (CTAs).

We present experimental analysis of cell death dynamics at photodynamic treatment in vitro and reveal features specific for different tumor localizations and individual patients. The analysis is based on monitoring of post-treatment changes in cellular morphology being recorded by means of digital holographic microscopy.

Understanding of the sequence of events that ensure invasiveness of malignant cells is important for prognostic purposes. The study of the cellular and molecular pathways in the metastatic process lays the foundation for further progress in the treatment of cancer patients.Purpose: a comparative analysis of in vitro migration and invasion of human solid tumor cells isolated from primary and metastatic lesions.Material and Methods. Cell cultures of skin melanoma (SM, n=29), renal cell cancer (RCC, n=2), colorectal cancer (CRC, n=1), soft tissue and bone sarcomas (STBS, n=39) isolated from solid human tumors were studied. Cell migration and invasion were assessed using xCelligence (ACEA Bioscience Inc., USA).Results. All solid tumor cell cultures demonstrated in vitro invasive potential (IP), which was 73.79 % for RCC; 53.16 % for SM; 43.96 % for STBS and 5.16 % for CRC. The rates of migration and invasion (SlopeInv) in STBS cells were higher than those in SM cells (39.33 and 25.3 μm/h (p<0.05), 95.32 and 59.82е-3, respectively (p<0.05). The differences in IP values depending on the origin of STBC cells (primary tumor, relapse, and metastasis) were revealed: 18.11 ± 3.05 %, 25.75 ± 5.57 %, 52.97 ± 5.64 %, respectively (p<0.05). We found a correlation between migration and invasion parameters of solid tumor cells and the expression of factors ensuring their mobility and affecting other cellular components of the tumor microenvironment, including cells of the immune system.Conclusion. The biologically «aggressive» phenotype of SM and STBS cells is associated with the expression of the cancer-testis genes, such as PRAME, PASD1, SSX1 and with the production of HB-EGF, IGFBP, PLGF, PECAM-1, FST, SCF, IL-8. These products can be considered as new targets for therapeutic technologies aimed at influencing metastatic disease.

The search of new predictor markers for disease outcomes and treatment efficacy is crucial for metastatic sarcoma, where prognosis is unfavourable and the choice of chemotherapy is limited.In our work we present the results of in vitro clonogenic abilities study of tumor cells connection with immunological parameters of peripheral blood and survival rate of patients with metastatic soft tissue sarcomas and bones sarcomas (STBS).

The paper presents an investigation of cell death dynamics and changes of cellular morphology induced by the intracellular generation of singlet oxygen as a result of photodynamic treatment. The response of kidney carcinoma, osteosarcoma, and skin melanoma cells to photodynamic treatment was analyzed through the changes in their thickness, average phase shift, and refractive index distribution. It was demonstrated that an irradiation dose of 42 J results in the necrosis of sarcoma and kidney carcinoma cells, although melanoma cells show no statistically significant changes in optical or morphological parameters.

We present a comparative analysis of photodynamic-treatment induced changes in optical parameters of cancer cells obtained from individual patients with three solid tumor localizations. Accumulation of photosensitizer inside living cells was validated using far-field fluorescence microscopy. Measurements of their optical characteristics were performed by means of digital holographic microscopy. The quantitative analysis of cell death dynamics performed by digital holographic microscopy was shown to be promising for investigation of cells resistivity to treatment. It was shown that both the photosensitizer accumulation and post-treatment dynamics of average phase shift may differ significantly in cell cultures obtained from different tumor localizations and different patients. Some of the cell cultures demonstrated very low or even no response to treatment.

Background: Recent advances in the cancer immunotherapy provide an opportunity to develop the therapeutic autologous dendritic cell vaccine for metastatic forms of soft tissues sarcomas (STS). Previous studies showed the possibility of developing delayed effects from this treatment. The aim of this study is to compare efficacy of the 2nd line therapy of STS patients with or without CaTeVac therapy during the whole systemic treatment. Methods: Seventy-four patients (pts.) were included since 2008 to 2017. All patients had stage III inoperable or stage IV STS and received 2nd line of systemic therapy. Group 1 received CaTeVac as adjuvant therapy or maintenance therapy after the 1st or 2nd line of systemic therapy or as monotherapy after at least 1 line of chemotherapy. Group 2 never received CaTeVac during treatment course. Patients in the groups were comparable by histologic types and previous treatment. Overall survival (OS) from the start of the 2nd line of therapy to death was assessed as efficacy measure. Results: Median OS of patients in group 1 was 24,4 mo (741 days, 95% CI, 509-973), versus 14.2 mo (431 days, 95% CI, 56-806) in group 2 (Log-Rank p = 0.019, Breslow p = 0.03). Relative risk of death (exp(B)) in group 1 was 0,358 (95% CI 0.171-0.751) in the Cox regression and was independent from and comparable to the treatment effect (exp(B) 0.254 (95% CI 0.133-0.484), p = 0.000011. Sex and age showed no impact on survival (p > 0.05). Conclusions: CaTeVac shows impact on overall survival and should be assessed in randomized clinical trials with OS endpoints. Legal entity responsible for the study: N.N. Petrov Research Institute of Oncology. Funding: Ministry of Health Russian Federation. Disclosure: All authors have declared no conflicts of interest.

Changes in optical and morphological parameters in three types of cancer cells from neoplastic material taken from patients and subjected to photodynamic treatment were monitored using digital holographic microscopy and tomography. The photoinduced cells death through necrosis pathway was clearly observed in soft tissue sarcoma and kidney carcinoma cells, while melanoma cells demonstrated no significant response to treatment.