Preparation of tumor cell lysates.
Tumor cell lysates were prepared from tissue samples after radical surgery or incisional biopsy.
A 1-3-cm3 tumor tissue sample was placed into a sterile tube, the adjacent (macroscopically unaltered) tissues were removed, and the sample was subjected to mechanical homogenization and four-freeze thaw cycle (-80oC and room temperature, respectively). Larger particles were removed by centrifugation at 266 x g and 24oC (room temperature) for 2 minutes. The lysate was filter-sterilized (filter pore diameter = 0.45 um) and the protein concentration of the lysate was measured using a NanoDrop 2000. The tumor cell lysate was divided into aliquots, frozen and stored -80oC.
DC preparation and characterization:
Mononuclear cells were isolated from the peripheral blood of the patients using Ficoll gradient centrifugation. The isolated peripheral blood mono- nuclear cells (PBMCs) were washed in RPMI- 1640 medium and centrifuged twice at 266 x g at 24oC (room temperature) for 10 min. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 80 ug/ml gentamycin, 100 ug/ ampicillin. Mononuclear cells (1-1.5×106 cells/ml) in RPMI-1640 complete medium supplemented with 10% FBS were placed into 150-cm2 (690 mL) culture flasks with vintage caps. The cells were allowed to adhere to the flask in CO2 incubator at 37OC and 100% humidity for 30 minutes. Viable non-adherent mononuclear cells (non-adherent PBMCs) were cultured in complete RPMI-1640 medium with partial media changes on days 3 and 5. The adherent cells were removed using a cell scraper and washed with RPMI-1640 medium.
The adherent cell fraction was used to generate mature antigen activated DCs. For this purpose, 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml IL-4 were added to the cells, which were cultured in a 75-cm2 (270mL) culture flask in complete RPMI-1640 medium supplemented with 10% FBS for 72 h to generate immature DCs. The tumor tissue lysate was added to immature DC for 24
hours. To obtain mature DCs, TNF-a was added to the fresh medium within 48 hours.
Clinical activity for patients with stage IV or progressive disease:
Among patients with stage IV disease, 2 patients remained stable for 6 months. 1 patient developed local progresion, distant foci were undetectable, and chemotherapy was not administered. 1 patient succumbed to disease progression (two injections were administered). Three lines of chemotherapy did not achieve positive response.
The treatment was generally well-tolerated. The patients were monitored by medical personnel for 24 h after injection to assess toxicity. The most common adverse event were flu-like symptoms, such as fever and fatigue, that did not require additional treatment or prolonged hospitalization. All symptoms spontaneously resolved without treatment after 2-3 h.
This result demonstrated that the investigated cellular immunotherapy for breast cancer is safe, reduces the risk of relapse and metastasis, and improves immunity by reducing the number of regulatory T cells. Therefore, this therapeutic strategy may represent a novel approach to combating distant metastases of breast cancer. Dendritic cell and Activated T cells in combination with other therapies is effective and prolonged life of patients with breast cancer.
Article Reference link: click here
Scientific article publishing date : 22/11/2019
Immucura identifier : BSC21_026EN