Protocol to construct humanized mice with adult CD34+ hematopoietic stem and progenitor cells.

Humanized mice, defined as mice with human immune systems, have become an emerging model to study human hematopoiesis, infectious disease, and cancer. Here, we describe the techniques to generate humanized NSGF6 mice using adult human CD34+ hematopoietic stem and progenitor cells (HSPCs). We describe steps for constructing and monitoring the engraftment of humanized mice. We then detail procedures for tissue processing and immunophenotyping by flow cytometry to evaluate the multilineage hematopoietic differentiation. For complete details on the use and execution of this protocol, please refer to Yu et al.1.

Engraftment of adult hematopoietic stem and progenitor cells in a novel model of humanized mice.

Pre-clinical use of humanized mice transplanted with CD34+ hematopoietic stem and progenitor cells (HSPCs) is limited by insufficient engraftment with adult non-mobilized HSPCs. Here, we developed a novel immunodeficient mice based on NOD-SCID-Il2γc-/- (NSG) mice to support long-term engraftment with human adult HSPCs. As both Flt3L and IL-6 are critical for many aspects of hematopoiesis, we knock-out mouse Flt3 and knock-in human IL6 gene. The resulting mice showed an increase in the availability of mouse Flt3L to human cells and a dose-dependent production of human IL-6 upon activation. Upon transplantation with low number of human HSPCs from adult bone marrow, these humanized mice demonstrated a significantly higher engraftment with multilineage differentiation of human lymphoid and myeloid cells, and tissue colonization at one year after adult HSPC transplant. Thus, these mice enable studies of human hematopoiesis and tissue colonization over time and may facilitate building autologous models for immuno-oncology studies.

Comprehensive single cell aging atlas of mammary tissues reveals shared epigenomic and transcriptomic signatures of aging and cancer.

Aging is the greatest risk factor for breast cancer; however, how age-related cellular and molecular events impact cancer initiation is unknown. We investigate how aging rewires transcriptomic and epigenomic programs of mouse mammary glands at single cell resolution, yielding a comprehensive resource for aging and cancer biology. Aged epithelial cells exhibit epigenetic and transcriptional changes in metabolic, pro-inflammatory, or cancer-associated genes. Aged stromal cells downregulate fibroblast marker genes and upregulate markers of senescence and cancer-associated fibroblasts. Among immune cells, distinct T cell subsets (Gzmk+, memory CD4+, γδ) and M2-like macrophages expand with age. Spatial transcriptomics reveal co-localization of aged immune and epithelial cells in situ. Lastly, transcriptional signatures of aging mammary cells are found in human breast tumors, suggesting mechanistic links between aging and cancer. Together, these data uncover that epithelial, immune, and stromal cells shift in proportions and cell identity, potentially impacting cell plasticity, aged microenvironment, and neoplasia risk.

Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility. For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1.

Long-term engraftment of adult hematopoietic progenitors in a novel model of humanized mice.

Pre-clinical use of humanized mice transplanted with CD34 + hematopoietic progenitor cells (HPCs) is limited by insufficient engraftment with adult HPCs. Here, we developed a novel immunodeficient mice based in NOD-SCID- Il2γc -/- (NSG) mice to support long-term engraftment with human adult HPCs and tissue colonization with human myeloid cells. As both Flt3L and IL-6 are critical for many aspects of hematopoiesis, we knock-out mouse Flt3 and knock-in human IL6 gene. The resulting mice showed an increase in the availability of mouse Flt3L to human cells, and a dose-dependent production of human IL-6 upon activation. Upon transplantation with low number of human HPCs from adult bone marrow, these humanized mice demonstrated a significantly higher engraftment with multilineage differentiation of human lymphoid and myeloid cells. Furthermore, higher frequencies of human lymphoid and myeloid cells were detected in tissues at one year after adult HPC transplant. Thus, these mice enable studies of human hematopoiesis and tissue colonization over time. Summary: Pre-clinical use of humanized mice is limited by insufficient engraftment with adult hematopoietic progenitor cells (HPCs). Here, we developed a novel immunodeficient mice which support long-term engraftment with adult bone marrow HPCs and facilitate building autologous models for immuno-oncology studies.

High-throughput bioprinting of the nasal epithelium using patient-derived nasal epithelial cells.

Progenitor human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models through biofabrication. However, this approach has limitations in terms of achieving the intricate three-dimensional (3D) structure of the natural nasal epithelium. 3D bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of progenitor hNECs ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4 weeks air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes, such as disease modeling, immunological studies, and drug screening.

Computational immune synapse analysis reveals T-cell interactions in distinct tumor microenvironments.

The tumor microenvironment (TME) and the cellular interactions within it can be critical to tumor progression and treatment response. Although technologies to generate multiplex images of the TME are advancing, the many ways in which TME imaging data can be mined to elucidate cellular interactions are only beginning to be realized. Here, we present a novel approach for multipronged computational immune synapse analysis (CISA) that reveals T-cell synaptic interactions from multiplex images. CISA enables automated discovery and quantification of immune synapse interactions based on the localization of proteins on cell membranes. We first demonstrate the ability of CISA to detect T-cell:APC (antigen presenting cell) synaptic interactions in two independent human melanoma imaging mass cytometry (IMC) tissue microarray datasets. We then generate melanoma histocytometry whole slide images and verify that CISA can detect similar interactions across data modalities. Interestingly, CISA histoctyometry analysis also reveals that T-cell:macrophage synapse formation is associated with T-cell proliferation. We next show the generality of CISA by extending it to breast cancer IMC images, finding that CISA quantifications of T-cell:B-cell synapses are predictive of improved patient survival. Our work demonstrates the biological and clinical significance of spatially resolving cell-cell synaptic interactions in the TME and provides a robust method to do so across imaging modalities and cancer types.

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment.

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.

Mouse models for immuno-oncology.

Realizing the clinical promise of cancer immunotherapy is hindered by gaps in our knowledge of in vivo mechanisms underlying treatment response as well as treatment limiting toxicity. Preclinical in vivo model systems and technologies are required to address these knowledge gaps and to surmount the challenges faced in the clinical application of immunotherapy. Mice are commonly used for basic and translational research to support development and testing of immune interventions, including for cancer. Here, we discuss the advantages and the limitations of current models as well as future developments.

Challenges and opportunities for modeling aging and cancer.

Age is among the main risk factors for cancer, and any cancer study in adults is faced with an aging tissue and organism. Yet, pre-clinical studies are carried out using young mice and are not able to address the impact of aging and associated comorbidities on disease biology and treatment outcomes. Here, we discuss the limitations of current mouse cancer models and suggest strategies for developing novel models to address these major gaps in knowledge and experimental approaches.

Multiplex immunofluorescence-guided laser capture microdissection for spatial transcriptomics of metastatic melanoma tissues.

We describe a pipeline for optimized and streamlined multiplexed immunofluorescence-guided laser capture microdissection allowing the harvest of individual cells based on their phenotype and tissue localization for transcriptomic analysis with next-generation RNA sequencing. Here, we analyze transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages, and melanoma cells in non-dissociated metastatic melanoma tissue. While this protocol is described for melanoma tissues, we successfully applied it to human tonsil, skin, and breast cancer tissues as well as mouse lung tissues. For complete details on the use and execution of this protocol, please refer to Martinek et al. (2022).

Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

Transcriptional profiling of macrophages in situ in metastatic melanoma reveals localization-dependent phenotypes and function.

Modulation of immune function at the tumor site could improve patient outcomes. Here, we analyze patient samples of metastatic melanoma, a tumor responsive to T cell-based therapies, and find that tumor-infiltrating T cells are primarily juxtaposed to CD14+ monocytes/macrophages rather than melanoma cells. Using immunofluorescence-guided laser capture microdissection, we analyze transcriptomes of CD3+ T cells, CD14 + monocytes/macrophages, and melanoma cells in non-dissociated tissue. Stromal CD14+ cells display a specific transcriptional signature distinct from CD14+ cells within tumor nests. This signature contains LY75, a gene linked with antigen capture and regulation of tolerance and immunity in dendritic cells (DCs). When applied to TCGA cohorts, this gene set can distinguish patients with significantly prolonged survival in metastatic cutaneous melanoma and other cancers. Thus, the stromal CD14+ cell signature represents a candidate biomarker and suggests that reprogramming of stromal macrophages to acquire DC function may offer a therapeutic opportunity for metastatic cancers.

Bronchial epithelium epithelial-mesenchymal plasticity forms aberrant basaloid-like cells in vitro.

Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. To elucidate the role of EMT in the development of aberrant basaloid cells from the bronchial epithelium, we mapped EMT-induced transcriptional changes at the population and single-cell levels. Human bronchial epithelial cells grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Comparison of submerged and ALI cultures revealed differential expression of 8,247 protein coding (PC) and 1,621 long noncoding RNA (lncRNA) genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,020 PC and 907 lncRNA genes. Although there was no evidence for fibroblast/myofibroblast conversion following EMT induction, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. The substantial transcriptional differences between submerged and ALI cultures highlight that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and an interactive browser (https://bronc-epi-in-vitro.cells.ucsc.edu/) of single-cell RNA-Seq data for further exploration of potential roles in the lung epithelium in health and lung disease.

A comprehensive long-read isoform analysis platform and sequencing resource for breast cancer.

Tumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is unknown. We developed a long-read (LR) RNA sequencing and analytical platform that identifies and annotates full-length isoforms and infers tumor-specific splicing events. Application of this platform to breast cancer samples identifies thousands of previously unannotated isoforms; ~30% affect protein coding exons and are predicted to alter protein localization and function. We performed extensive cross-validation with -omics datasets to support transcription and translation of novel isoforms. We identified 3059 breast tumor­specific splicing events, including 35 that are significantly associated with patient survival. Of these, 21 are absent from GENCODE and 10 are enriched in specific breast cancer subtypes. Together, our results demonstrate the complexity, cancer subtype specificity, and clinical relevance of previously unidentified isoforms and splicing events in breast cancer that are only annotatable by LR-seq and provide a rich resource of immuno-oncology therapeutic targets.

Developing and validating model systems for immuno-oncology.

Owing to clinical success of immune-checkpoint blockade, immunotherapy is becoming a cornerstone of modern oncology, and immuno-oncology is at the forefront of basic cancer research. This commentary outlines future opportunities for immuno-oncology modeling.

Human KIT+ myeloid cells facilitate visceral metastasis by melanoma.

Metastasis of melanoma significantly worsens prognosis; thus, therapeutic interventions that prevent metastasis could improve patient outcomes. Here, we show using humanized mice that colonization of distant visceral organs with melanoma is dependent upon a human CD33+CD11b+CD117+ progenitor cell subset comprising <4% of the human CD45+ leukocytes. Metastatic tumor-infiltrating CD33+ cells from patients and humanized (h)NSG-SGM3 mice showed converging transcriptional profiles. Single-cell RNA-seq analysis identified a gene signature of a KIT/CD117-expressing CD33+ subset that correlated with decreased overall survival in a TCGA melanoma cohort. Thus, human CD33+CD11b+CD117+ myeloid cells represent a novel candidate biomarker as well as a therapeutic target for metastatic melanoma.

Definition of erythroid cell-positive blood transcriptome phenotypes associated with severe respiratory syncytial virus infection.

Biomarkers to assess the risk of developing severe respiratory syncytial virus (RSV) infection are needed. We conducted a meta-analysis of 490 unique profiles from six public RSV blood transcriptome datasets. A repertoire of 382 well-characterized transcriptional modules was used to define dominant host responses to RSV infection. The consolidated RSV cohort was stratified according to four traits: "interferon response" (IFN), "neutrophil-driven inflammation" (Infl), "cell cycle" (CC), and "erythrocytes" (Ery). We identified eight prevalent blood transcriptome phenotypes, of which three Ery+ phenotypes comprised higher proportions of patients requiring intensive care. This finding confirms on a larger scale data from one of our earlier reports describing an association between an erythrocyte signature and RSV disease severity. Further contextual interpretation made it possible to associate this signature with immunosuppressive states (late stage cancer, pharmacological immunosuppression), and with a population of fetal glycophorin A+ erythroid precursors. Furthermore, we posit that this erythrocyte cell signature may be linked to a population of immunosuppressive erythroid cells previously described in the literature, and that overabundance of this cell population in RSV patients may underlie progression to severe disease. These findings outline potential priority areas for biomarker development and investigations into the immune biology of RSV infection. The approach that we developed and employed here should also permit to delineate prevalent blood transcriptome phenotypes in other settings.

Techniques for the generation of humanized mouse models for immuno-oncology.

Mouse models of human cancer have been used extensively to circumvent the complexity in human patients. However, murine models often inadequately recapitulate the human cancer-immune interface partly due to important differences between mouse and human immune systems. Immunodeficient mice when transplanted with CD34+ hematopoietic progenitor cells (HPCs) develop multilineage human immune cells. While there remain limitations, efforts have been made to improve the function of human immune system. Thus, humanized mice, defined as mice with human immune system, have become an emerging model to study human cancers. Humanized mouse models have been used for various areas of cancer research including adoptive transfer of chimeric antigen receptor (CAR)-modified T cells, neoantigen vaccination to increase T cell repertoire and reprograming tumor microenvironment. Here, we describe the essential techniques to generate humanized mouse models for immuno-oncology studies.

Interplay between dendritic cells and cancer cells.

Dendritic cells (DCs) orchestrate a repertoire of immune responses that bring about resistance to infection and tolerance to self. Cancers can exploit DCs to evade immunity, but DCs also can generate resistance to cancer. Owing to their capacity to capture, process, and present antigens to naïve T cells, thereby launching adaptive immunity, DCs are poised to play a critical role in cancer recognition and rejection. As such, DCs represent a solution for the expansion and infiltration of T cells with tumor-rejecting properties. Indeed, clinical responses to checkpoint blockade, such as anti-PD-1, are linked to the presence of T cell immunity to cancer-specific antigens. However, only a fraction of patients has clinical benefit. Unraveling the molecular pathways controlling DC-cancer interplay will therefore pave the way for identifying new targets for therapy that overcome limitations of current treatments and promote long-term cancer control.