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Malignant pleural effusion (MPE) is a common complication of lung adenocarcinoma (LADC) which is associated with a dismal prognosis. We investigated the prognostic role of PD-L1 and other immunomodulators expression in the immune compartment of MPE immune composition. MPE cytologic cell blocks of 83 LADC patients were analysed for the mRNA expression of 770 cancer-immune genes by the NanoString nCounter platform. The expression of relevant immune cell lineage markers was validated by immunohistochemistry (IHC) using quantitative pathology. The mRNA immune profiling identified four MPE patient clusters (C). C1/2 (adaptive+, hot) showed better overall survival (OS) than C3/4 (adaptive-, cold). Additionally, cold immunity profiles (adaptive-), C4 (innate+) were associated with worse OS than C3 (innate-). High PD-L1 expression was linked to the regulation of T cell activation and interferon signalling pathways. Genes of pattern recognition receptor and type I interferon signalling pathways were specifically upregulated in the long-survival (≥90 days) patient group. Moreover, immunomodulators were co-activated and highly expressed in hot adaptive immunity patient clusters, whereas CD274 (PD-L1), TNFRSF9 (4-1BB), VEGFA (VEGF-A) and CD276 (B7-H3) were upregulated in the groups referred as cold. The patient cluster, age and PD-L1 expression were independent prognosticators for LADC MPE patients (p-value < 0.05). Our study sheds light on the variances of immune contexture regarding different PD-L1 expression and survival conditions. It revealed four distinct prognostic patient clusters with specific immune cell components and immunomodulator expression profiles, which, collectively, is supportive for future therapeutic and prognosis for cancer management.

Background Malignant serous effusion (MSE) denotes a manifestation of metastatic disease with typical high concentrations of both cancer and immune cells, making them an ideal resource for in vitro cytologic studies. Hence, the aim of the study was to investigate the features of 2D and 3D MSE culture systems as well as their feasibilities for in vitro drug screening. Methods Pleural and peritoneal effusions from 8 patients were collected and processed for 2D monolayer and 3D hanging drop cell culture into GravityPLUS™ plates. Representative markers for cell components, proliferation rate and tumour classification were investigated by immunohistochemistry, followed by absolute quantification using a digitalised image analysis approach. Further, we implemented another 3D cell culture model based on a low attachment method for in vitro drug sensitivity testing of carboplatin, pemetrexed and pembrolizumab for 5 patients. Results Monolayer cell culture was favourable for the growth of mesothelial cells, while hanging drop culture in GravityPLUS™ plates showed better ability for preserving cancer cells, inducing positive diagnostic markers expression and restraining the growth of mesothelial cells. For in vitro drug testing, MSE from five patients presented various drug sensitivities, and one case showed strong response to PD-1 checkpoint inhibition (pembrolizumab). For some patients, the application of combinatorial drugs had better therapeutic responses compared to monotherapy. Conclusions Digitalised quantification of data offers a better understanding of different MSE culture models. More importantly, the proposed platforms are practical and amenable for performing in vitro chemo-/immunotherapeutic drug testing by using routine cytologic MSE in a personalised manner. Next to cell blocks, our work demonstrates the prognostic and predictive value of cytologic effusion samples.