Evaluation of tumor response to immune checkpoint inhibitors by a 3D immunotumoroid model.

Background: Only 20 percent of renal and bladder cancer patients will show a significant response to immune checkpoint inhibitor (ICI) therapy, and no test currently available accurately predicts ICI response. Methods: We developed an "immunotumoroid" cell model system that recapitulates the tumor, its microenvironment, and necessary immune system components in patient-derived spheroids to enable ex vivo assessment of tumor response to ICI therapy. Immunotumoroids were developed from surgically resected renal cell carcinomas and bladder carcinomas selected for high tumor-infiltrating lymphocytes (TILs) and survived more than a month without media exchange. Immunohistochemistry was used to detect immune and non-immune cells in cryopreserved source tumors and the resulting immunotumoroids. Immunotumoroid response to ICIs (nivolumab, pembrolizumab, and durvalumab) and chemotherapy (cisplatin, gemcitabine, and paclitaxel) was monitored in real-time with Cytotox Red staining in an Incucyte device, and the immunotumoroid response was compared to retrospective clinical drug responses. Results: Six of the 13 cases tested grew viable immunotumoroid models, with failed cases attributed to extensive tumor tissue necrosis or excess lymphocytes preventing spheroid formation. One successfully cultured case was excluded from the study due to low TIL infiltration (<5%) in the primary tumor sample. The five remaining models contained immune cells (CD4+ and CD8+ T cells, and macrophages), non-immune cells (fibroblasts), and tumor cells. Chemotherapy and ICI drugs were tested in immunotumoroids from 5 cases and compared to clinical outcomes where data was available. Four/five models showed cell killing in response to chemotherapy and two/five showed sensitivity to ICI. In three cases, the immunotumoroid model accurately predicted the patient's clinical response or non-response to ICIs or chemotherapy. Conclusion: Our immunotumoroid model replicated the multicellular nature of the tumor microenvironment sufficiently for preclinical ICI screening. This model could enable valuable insights into the complex interactions between cancer cells, the immune system, and the microenvironment. This is a feasibility study on a small number of cases, and additional studies with larger case numbers are required including correlation with clinical response.

Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation.

Ex vivo drug screening is a potentially powerful tool for the future of cancer care, but the accuracy of results is contingent on the culture model. Both monolayer (2D) and spheroid (3D) culture systems offer advantages, but given the differences in mechanical environment, we hypothesized that that the suitability of one system over another would be critical for screening drugs with mechanical targets in mechanical tissues. HCC827 lung adenocarcinoma cells were challenged with EGFR tyrosine kinase inhibitors in monolayer and spheroid culture. RNA sequencing was performed on cells in both conditions to assess culture-induced transcriptional changes that could account for differences in drug response and differences in EGFR expression detected by immunostain. A microRNA microarray was performed to assess culture-induced differences in regulation of microRNA, and the impact of miR-146a-5p on drug response was verified by inhibition. Results were confirmed in human lung adenocarcinoma tissue. HCC827 spheroids were resistant to erlotinib and gefitinib, but significantly more sensitive in 2D culture. RNA-seq and immunostaining show a discrepancy in EGFR transcript and protein expression between the two conditions, which we attribute to miR-146a-5p. This microRNA targets EGFR and is differentially expressed between 2D and 3D culture. Inhibition of miR-146a-5p significantly increased erlotinib cytotoxicity, but validation in patient-derived spheroids suggests that the effect may be mutation-specific. Analysis of RNA-seq data suggests that cells in 2D culture become highly dependent on EGFR signaling to drive proliferation and cell spreading, resulting in a misleading level of sensitivity to EGFR TKIs, while the same cells in spheroid culture retain microRNA-driven EGFR feedback regulation that leaves them less vulnerable to EGFR inhibition. These findings underscore the need for close scrutiny of culture-induced effects on drug target regulation in model design for ex vivo drug screening.

Experimental estimation of entanglement at the quantum limit.

Entanglement is the central resource of quantum information processing and the precise characterization of entangled states is a crucial issue for the development of quantum technologies. This leads to the necessity of a precise, experimental feasible measure of entanglement. Nevertheless, such measurements are limited both from experimental uncertainties and intrinsic quantum bounds. Here we present an experiment where the amount of entanglement of a family of two-qubit mixed photon states is estimated with the ultimate precision allowed by quantum mechanics.