The evaluation of the response to treatments will be performed when PDTO have reached a diameter of 150 μm in « PDTO treatment medium », corresponding to the PDTO culture medium lacking N-Acetylcysteine, Y-27632 and primocin.
PDTO will be collected, resuspended in 2% extracellular matrix/PDTO culture medium and then platted in white and clear bottom 96-well plates previously coated with a 1:1 volume mix of PDTO treatment medium with extracellular matrix. In the case of evaluation of the response to radiotherapy, PDTO will be before irradiated using the CellRad System (FAXITRON Bioptics). In the case of evaluation of the response to chemotherapy or PARP inhibitors, drugs are prepared in 2% extracellular matrix/PDTO culture medium and added 1 hour after PDTO have been plated.
In the case of evaluation of the response to immunotherapies, PDTO will be co-cultured with PDTO specific T cells previously generated (see co-culture of PDTO with immune cells) at a 5:1 ratio. Treatments (such as Nivolumab or Pembrolizumab) will be added directly in the co-culture. A condition containing an MHC-I blocking antibody will be added to control for antigen specific killing.
PDTO morphology will be monitored by taking images during the required time using Incucyte S3 (Sartorius). At the endpoint, PDTO response will be assessed using CellTiter-Glo 3D cell viability assay (Promega) according to the manufacturer’s instruction and luminescence will be measured using GloMax Discover GM3000 (Promega) with the associated software. Results will be normalized to the control condition. IC50 will be calculated with GraphPad software. The ability of T cells to recognize and induce lysis of PDTO will be monitored via analysis of caspase 3 cleavage within PDTO and visualization of LAMP-1 on the membrane of CD8+ T cells.
The treatment response of the PDTO will be finally compared to the clinical response (PFS/DFS/OS) of the patient from whom they are derived in order to validate the predictive value of this model for HNSCC.
PDTO will be collected, resuspended in 2% extracellular matrix/PDTO culture medium and then platted in white and clear bottom 96-well plates previously coated with a 1:1 volume mix of PDTO treatment medium with extracellular matrix. In the case of evaluation of the response to radiotherapy, PDTO will be before irradiated using the CellRad System (FAXITRON Bioptics). In the case of evaluation of the response to chemotherapy or PARP inhibitors, drugs are prepared in 2% extracellular matrix/PDTO culture medium and added 1 hour after PDTO have been plated.
In the case of evaluation of the response to immunotherapies, PDTO will be co-cultured with PDTO specific T cells previously generated (see co-culture of PDTO with immune cells) at a 5:1 ratio. Treatments (such as Nivolumab or Pembrolizumab) will be added directly in the co-culture. A condition containing an MHC-I blocking antibody will be added to control for antigen specific killing.
PDTO morphology will be monitored by taking images during the required time using Incucyte S3 (Sartorius). At the endpoint, PDTO response will be assessed using CellTiter-Glo 3D cell viability assay (Promega) according to the manufacturer’s instruction and luminescence will be measured using GloMax Discover GM3000 (Promega) with the associated software. Results will be normalized to the control condition. IC50 will be calculated with GraphPad software. The ability of T cells to recognize and induce lysis of PDTO will be monitored via analysis of caspase 3 cleavage within PDTO and visualization of LAMP-1 on the membrane of CD8+ T cells.
The treatment response of the PDTO will be finally compared to the clinical response (PFS/DFS/OS) of the patient from whom they are derived in order to validate the predictive value of this model for HNSCC.
Free full text: Click here
