Beside standard CRISPR editing (Knock-In and -Out) our lab utilize also the CRISPR interference approach where we either apply activation (CRISPRa) or inhibition (CRISPRi) to explore how epigenetically regulated genes support the development or repression of cancer. These techniques have endless opportunities and thus we have multiple projects ongoing that use these techniques not only in cancer, but also in other aspects of immune regulation for different modalities.

Lipid nanoparticles (LNPs) are small vesicles capable of transfecting a broad range of drug molecules into cells either during in vitro cultures or in more complexed in vivo settings. They have been highly popularized recently, by being utilized in many SARS-CoV-2 vaccine approaches. Our LNPs are lipid droplets consisting of very specific lipid formulations containing cargo (ranging from mRNA, protein, and small molecule drugs). By conjugating antibodies to the surface of the LNPs we aim to ensure cell specific uptake, to ensure more specific effects. Using LNPs lets us move between in vitro and in vivo work more directly and allows us to bridge the gap between lab work and therapeutics.

One crucial aspect of the research in the Laboratory for Cancer, Immunity, and Inflammation is the utilization of in vivo disease models. These models serve as invaluable tools that allow us to study disease development, progression, and response to therapies in a complex and dynamic biological context.

For example in vivo cancer models involve the transplantation or induction of cancer cells or tumors into living organisms, such as mice. These models closely mimic the physiological and microenvironmental conditions encountered in human cancers, providing us with a more realistic representation of the disease and its interactions with the immune system and inflammatory processes. Our laboratory utilizes a wide range of in vivo cancer models, including human cancer cell lines xenografts (PDX) and syngeneic murine tumor models. For the syngeneic tumor models, we implant tumor cells from the same species into immunocompetent mice, allowing us to investigate immune responses against the tumor and evaluate immunotherapeutic strategies. Therapeutic testing includes: chemotherapy, checkpoint inhibitors, radiation, immuno-agonists, small molecules, adaptive cell therapies, and cytokine therapy.

Overall, our use of mouse models provide us with a platform to test novel therapeutic approaches, study the mechanisms of action of immunotherapies, evaluate drug candidates, and identify potential biomarkers for predicting treatment responses.

Tumor organoids are three-dimensional (3D) cell cultures that mimic the structural and functional characteristics of tumors more accurately than traditional two-dimensional (2D) cell cultures.

In these models, tumor cells are isolated from patient samples or genetically engineered to reflect specific cancer types. These cells are then embedded in a matrix that provides support and mimics the natural extracellular environment. Over time, these cells self-organize and proliferate, forming complex structures resembling the architecture and heterogeneity observed in actual tumors.

We have established various patient-derived models for lung cancer. We use them to study interactions with various immune cells under inflammatory conditions. This enables us to investigate the dynamic interplay between cancer cells and their surrounding microenvironment, in very controlled settings.  

Through our work with tumor organoids, we aim to advance our understanding of cancer, uncover new therapeutic avenues, and contribute to the development of personalized treatment strategies that can improve patient outcomes and ultimately lead to more effective and targeted therapies for cancer.

Spectral flow cytometry is an advanced flow cytometry technique that offers significant advantages over conventional flow cytometry methods. Instead of relying on traditional optical filters to detect fluorescent signals, spectral flow cytometry measures the entire emission spectrum of each fluorochrome in a single measurement. By utilizing spectral unmixing algorithms and sophisticated data analysis software, spectral flow cytometry can accurately distinguish overlapping spectra and deconvolute the signals from multiple fluorochromes. This allows for increased multiplexing capacity while maintaining the high throughput property of conventional flow cytometry. This makes spectral flow cytometry ideal for in-dept analysis of immune cells including analysis of rare immune cell subsets.

Our lab has recently developed and validated a 37-marker immune system panel for human samples and a similar 36-marker panel for murine species.

Meso Scale Discovery (MSD) ELISA is an advanced immunoassay technology that offers exceptional sensitivity and dynamic range for detecting proteins in biological samples. Unlike traditional ELISA, MSD ELISA utilizes electrochemiluminescence detection, resulting in enhanced accuracy and minimal interference. This technology is particularly valuable in biomarker research enabling researchers and clinicians to measure multiple analytes in a single sample using minimal sample volume. With its high-throughput capabilities and robust performance, Meso Scale ELISA has become a pivotal tool in unraveling complex molecular insights in various scientific domains.

Plasmacytoid dendritic cells (pDCs) are multifunctional immune cells that have a role in both the innate and adaptive immune system and are known for producing large amounts of type I interferons in response to viral infections. Their functions include cytokine production, antigen presentation, and cytotoxicity, and their potential as an immunotherapy for cancer and infectious disease is being explored. However, broad application of these cells is challenged by their low numbers in the blood representing only 0.1-0.5% of human PBMCs and by their low viability during ex vivo culturing. 

Our group has previously developed an effective approach for producing pDCs in vitro from CD34+ hematopoietic stem and progenitor cells (HSPC-pDCs), which provide an attainable source of pDCs for therapeutic purposes. HSPC-pDCs present pDC characteristics and functions, and like naturally occurring pDCs they exhibit phenotypic heterogeneity based on the expression of the pDC-associated cell surface markers. We decipher the phenotypic variability displayed by HSPC-pDCs by characterizing different subsets of pDCs. We also investigate the possibility of rerouting the cell fate of CD34+ HSPC during pDC specification by controlling gene expression of key master transcription factors (TFs). Furthermore, we functionally characterize different pDC subsets by applying CRISPR manipulation during differentiation thereby generating gene-specific knockout/in pDCs. Such pDC products are used to explore the anti-tumoral funtionality of pDCs in various in vitro settings.

• Western blotting
• Multiplex qPCR
• single cell/nuclei RNAseq
• Multiplex ELISA
• Generation and manipulation of primary human immune cells