Immunophenotyping with (phospho)protein profiling and fluorescent cell barcoding for single-cell signaling analysis and biomarker discovery.

The microenvironment of hematologic cancers contributes to tumor cell survival and proliferation, as well as treatment resistance. Understanding tumor- and drug-induced changes to the immune cell composition and functionality is therefore critical for implementing optimal treatment strategies and for the development of novel cancer therapies. The liquid nature of peripheral blood makes this organ uniquely suited for single-cell studies by flow cytometry. (Phospho)protein profiles detected by flow cytometry analyses have been shown to correlate with ex vivo drug sensitivity and to predict treatment outcomes in hematologic cancers, demonstrating that this method is suitable for pre-clinical studies. Here, we present a flow cytometry protocol that combines multi-parameter immunophenotyping with single-cell (phospho)protein profiling. The protocol makes use of fluorescent cell barcoding, which means that multiple cell samples, either collected from different donors or exposed to different treatment conditions, can be combined and analyzed as one experiment. This reduces variability between samples, increases the throughput of the experiment, and lowers experimental costs. This protocol may serve as a guide for the use and further development of assays to study immunophenotype and cell signaling at single-cell resolution in normal and malignant cells. The read-outs may provide biological insight into cancer pathogenesis, identify novel drug targets, and ultimately serve as a biomarker to guide clinical decision-making.

Standardized assays to monitor drug sensitivity in hematologic cancers.

The principle of drug sensitivity testing is to expose cancer cells to a library of different drugs and measure its effects on cell viability. Recent technological advances, continuous approval of targeted therapies, and improved cell culture protocols have enhanced the precision and clinical relevance of such screens. Indeed, drug sensitivity testing has proven diagnostically valuable for patients with advanced hematologic cancers. However, different cell types behave differently in culture and therefore require optimized drug screening protocols to ensure that their ex vivo drug sensitivity accurately reflects in vivo drug responses. For example, primary chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells require unique microenvironmental stimuli to survive in culture, while this is less the case for acute myeloid leukemia (AML) cells. Here, we present our optimized and validated protocols for culturing and drug screening of primary cells from AML, CLL, and MM patients, and a generic protocol for cell line models. We also discuss drug library designs, reproducibility, and quality controls. We envision that these protocols may serve as community guidelines for the use and interpretation of assays to monitor drug sensitivity in hematologic cancers and thus contribute to standardization. The read-outs may provide insight into tumor biology, identify or confirm treatment resistance and sensitivity in real time, and ultimately guide clinical decision-making.

A tumor microenvironment model of chronic lymphocytic leukemia enables drug sensitivity testing to guide precision medicine.

The microenvironment of chronic lymphocytic leukemia (CLL) cells in lymph nodes, spleen, and bone marrow provides survival, proliferation, and drug resistance signals. Therapies need to be effective in these compartments, and pre-clinical models of CLL that are used to test drug sensitivity must mimic the tumor microenvironment to reflect clinical responses. Ex vivo models have been developed that capture individual or multiple aspects of the CLL microenvironment, but they are not necessarily compatible with high-throughput drug screens. Here, we report on a model that has reasonable associated costs, can be handled in a regularly equipped cell lab, and is compatible with ex vivo functional assays including drug sensitivity screens. The CLL cells are cultured with fibroblasts that express the ligands APRIL, BAFF and CD40L for 24 h. The transient co-culture was shown to support survival of primary CLL cells for at least 13 days, and mimic in vivo drug resistance signals. Ex vivo sensitivity and resistance to the Bcl-2 antagonist venetoclax correlated with in vivo responses. The assay was used to identify treatment vulnerabilities and guide precision medicine for a patient with relapsed CLL. Taken together, the presented CLL microenvironment model enables clinical implementation of functional precision medicine in CLL.

The Norwegian childhood cancer biobank.

BACKGROUND: The rapidly expanding era of "omics" research is highly dependent on the availability of quality-proven biological material, especially for rare conditions such as pediatric malignancies. Professional biobanks provide such material, focusing on standardized collection and handling procedures, distinctive quality measurements, traceability of storage conditions, and accessibility. For pediatric malignancies, traditional tumor biobanking is challenging due to the rareness and limited amount of tissue and blood samples. The higher molecular heterogeneity, lower mutation rates, and unique genomic landscapes, however, renders biobanking of this tissue even more crucial. AIM: The aim of this study was to test and establish methods for a prospective and centralized biobank for infants, children, and adolescents up to 18 years of age diagnosed with cancer in Norway. METHODS: Obtain judicial and ethical approvals and administration through a consortium, steering committee, and advisory board. Develop pipelines including SOPs for all aspects in the biobank process, including collection, processing and storing of samples and data, as well of quality controlling, safeguarding, distributing, and transport. RESULTS: The childhood cancer biobanking started at Oslo University Hospital in March 2017 and was from 2019 run as a national Norwegian Childhood Cancer Biobank. Informed consent and biological samples are collected regionally and stored centrally. Approximately 12 000 samples from 510 patients and have been included by January 1, 2021, representing a 96% consent and participation rate among our newly diagnosed patients. CONCLUSION: A well-functioning nationwide collection and centralized biobank with standardized procedures and national storage for pediatric malignancies has been established with a high acceptance among families.

An in vitro assay for biomarker discovery and dose prediction applied to ibrutinib plus venetoclax treatment of CLL.

Recently, several small molecule drugs were approved for treatment of chronic lymphocytic leukemia (CLL), significantly improving patient management. However, knowledge about how to combine these therapies for optimal effects and what patients will best benefit from them is lacking. Here, we show that drug synergies can be identified by single cell signaling analyses. We investigated the effects of idelalisib, ibrutinib, and venetoclax on 35 protein epitopes by phospho flow in CLL cells. The activity of proteins in the B-cell receptor signalosome and the phosphatidylinositol 3-kinase pathway were altered upon drug exposure. Combined treatment with ibrutinib and venetoclax give promising results in clinical studies and we show that this combination exerted synergistic inhibitory effects on cell signaling and cell viability. Cell viability was monitored by flow cytometry and with independent drug sensitivity screens. Our analyses indicate that the standard dosages of ibrutinib and venetoclax can be lowered without loss of efficacy, potentially reducing drug costs, and toxicities. Observed correlation between signaling and viability indicates that signaling molecules could serve as biomarkers to predict response to therapy. We suggest that phospho flow should be considered as a novel approach for dose and synergy prediction in a precision medicine setting for CLL.

Cryopreservation of primary B cells minimally influences their signaling responses.

Phospho flow is a powerful approach to detect cell signaling aberrations, identify biomarkers and assess pharmacodynamics, and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here, B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions, fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes.

Single cell profiling of phospho-protein levels in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has a high incidence and a steeply growing prevalence in the Western world. The heterogeneity of the disease necessitates individual mapping of biology and predicted drug response in each patient as basis for administration of tailored treatments. Cell signaling aberrations may serve as biological indicators for suitable therapy. By applying phospho-specific flow cytometry, we mapped basal and induced phosphorylation levels of 20 phospho-epitopes on proteins relevant to B-cell signaling in B cells from 22 CLL patients and 25 normal controls. The signaling response of the cytostatic drugs fludarabine, doxorubicin and vincristine was also investigated. CLL cells exerted similar or lower basal phosphorylation levels compared to normal B cells, with the exception of STAT3 (pY705) which was increased. Interestingly, STAT3 inhibitors normalized the STAT3 (pY705) level and reduced cell viability. Vincristine treatment significantly modulated phosphorylation levels in CLL cells, while no effect was observed in controls or after fludarabine or doxorubicin treatment. After BCR stimulation, CLL cells showed a tendency towards impaired phosphorylation levels, significant for several of the analyzed proteins. However, the level of Akt (pS473) was more potently induced in IgHV unmutated CLL (UM-CLL) patient samples and was significantly higher than in M-CLL samples. Importantly, the PI3Kδ inhibitor idelalisib potently reversed the effect of anti-IgM on Akt (pS473). Thus, signaling aberrations could be identified by phosphoflow cytometry and aberrant signaling could be normalized by small molecule drugs. This approach can identify relevant drug targets as well as drug effects in the individual patient.