A path to translation: How 3D patient tumor avatars enable next generation precision oncology.

3D patient tumor avatars (3D-PTAs) hold promise for next-generation precision medicine. Here, we describe the benefits and challenges of 3D-PTA technologies and necessary future steps to realize their potential for clinical decision making. 3D-PTAs require standardization criteria and prospective trials to establish clinical benefits. Innovative trial designs that combine omics and 3D-PTA readouts may lead to more accurate clinical predictors, and an integrated platform that combines diagnostic and therapeutic development will accelerate new treatments for patients with refractory disease.

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).

Single-Cell Transcriptome Analysis Defines Expression of Kabuki Syndrome-Associated KMT2D Targets and Interacting Partners.

Objectives. Kabuki syndrome (KS) is a rare genetic disorder characterized by developmental delay, retarded growth, and cardiac, gastrointestinal, neurocognitive, renal, craniofacial, dental, and skeletal defects. KS is caused by mutations in the genes encoding histone H3 lysine 4 methyltransferase (KMT2D) and histone H3 lysine 27 demethylase (KDM6A), which are core components of the complex of proteins associated with histone H3 lysine 4 methyltransferase SET1 (SET1/COMPASS). Using single-cell RNA data, we examined the expression profiles of Kmt2d and Kdm6a in the mouse dental pulp. In the incisor pulp, Kmt2d and Kdm6a colocalize with other genes of the SET1/COMPASS complex comprised of the WD-repeat protein 5 gene (Wdr5), the retinoblastoma-binding protein 5 gene (Rbbp5), absent, small, and homeotic 2-like protein-encoding gene (Ash2l), nuclear receptor cofactor 6 gene (Ncoa6), and Pax-interacting protein 1 gene (Ptip1). In addition, we found that Kmt2d and Kdm6a coexpress with the downstream target genes of the Wingless and Integrated (WNT) and sonic hedgehog signaling pathways in mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation. Taken together, our results suggest an essential role of KMT2D and KDK6A in directing lineage-specific gene expression during differentiation of MSCs.

Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues.

Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus. It affects many women during their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, which limits early diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared them to matched eutopic endometrium, unaffected endometrium and organoids derived from these tissues, generating data on over 122,000 cells across 14 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell specific to the peritoneal lesions, with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesion microenvironments and an unreported progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment that represents a comprehensive cell atlas of the disease in individuals undergoing hormonal treatment, providing essential information for future therapeutics and diagnostics.

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.

Single-cell transcriptome analysis defines mesenchymal stromal cells in the mouse incisor dental pulp.

The dental pulp is known to be highly heterogenous, comprising distinct cell types including mesenchymal stromal cells (MSCs), which represent neural-crest-derived cells with the ability to differentiate into multiple cell lineages. However, the cellular heterogeneity and the transcriptome signature of different cell clusters within the dental pulp remain to be established. To better understand discrete cell types, we applied a single-cell RNA sequencing strategy to establish the RNA expression profiles of individual dental pulp cells from 5- to 6-day-old mouse incisors. Our study revealed distinct subclasses of cells representing osteoblast, odontoblast, endothelial, pancreatic, neuronal, immune, pericyte and ameloblast lineages. Collectively, our research demonstrates the complexity and diversity of cell subclasses within the incisor dental pulp, thus providing a foundation for uncovering the molecular processes that govern cell fate decisions and lineage commitment in dental pulp-derived MSCs.

Single-cell multimodal glioma analyses identify epigenetic regulators of cellular plasticity and environmental stress response.

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.

Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies.

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.

Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities.

Despite molecular and clinical heterogeneity, small cell lung cancer (SCLC) is treated as a single entity with predictably poor results. Using tumor expression data and non-negative matrix factorization, we identify four SCLC subtypes defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures accompanied by an Inflamed gene signature (SCLC-A, N, P, and I, respectively). SCLC-I experiences the greatest benefit from the addition of immunotherapy to chemotherapy, while the other subtypes each have distinct vulnerabilities, including to inhibitors of PARP, Aurora kinases, or BCL-2. Cisplatin treatment of SCLC-A patient-derived xenografts induces intratumoral shifts toward SCLC-I, supporting subtype switching as a mechanism of acquired platinum resistance. We propose that matching baseline tumor subtype to therapy, as well as manipulating subtype switching on therapy, may enhance depth and duration of response for SCLC patients.

Antibody targeting of B7-H4 enhances the immune response in urothelial carcinoma.

Patients with locally advanced and metastatic urothelial carcinoma have a low survival rate (median 15.7 months, 13.1-17.8), with only a 23% response rate to monotherapy treatment with anti-PDL1 checkpoint immunotherapy. To identify new therapeutic targets, we profiled the immune regulatory signatures during murine cancer development using the BBN carcinogen and identified an increase in the expression of the T cell inhibitory protein B7-H4 (VTCN1, B7S1, B7X). B7-H4 expression temporally correlated with decreased lymphocyte infiltration. While the increase in B7-H4 expression within the bladder by CD11b+ monocytes is shared with human cancers, B7-H4 expression has not been previously identified in other murine cancer models. Higher expression of B7-H4 was associated with worse survival in muscle-invasive bladder cancer in humans, and increased B7-H4 expression was identified in luminal and luminal-papillary subtypes of bladder cancer. Evaluation of B7-H4 by single-cell RNA-Seq and immune mass cytometry of human bladder tumors found that B7-H4 is expressed in both the epithelium of urothelial carcinoma and CD68+ macrophages within the tumor. To investigate the function of B7-H4, treatment of human monocyte and T cell co-cultures with a B7-H4 blocking antibody resulted in enhanced IFN-γ secretion by CD4+ and CD8+ T cells. Additionally, anti-B7-H4 antibody treatment of BBN-carcinogen bladder cancers resulted in decreased tumor size, increased CD8+ T cell infiltration within the bladder, and a complimentary decrease in tumor-infiltrating T regulatory cells (Tregs). Furthermore, treatment with a combination of anti-PD-1 and anti-B7-H4 antibodies resulted in a significant reduction in tumor stage, a reduction in tumor size, and an increased level of tumor necrosis. These findings suggest that antibodies targeting B7-H4 may be a viable strategy for bladder cancers unresponsive to PD-1 checkpoint inhibitors.

Transplanting cells from old but not young donors causes physical dysfunction in older recipients.

Adipose-derived mesenchymal stem cell (ADSC)-based regenerative therapies have shown potential for use in many chronic diseases. Aging diminishes stem cell regenerative potential, yet it is unknown whether stem cells from aged donors cause adverse effects in recipients. ADSCs can be obtained using minimally invasive approaches and possess low immunogenicity. Nevertheless, we found that transplanting ADSCs from old donors, but not those from young donors, induces physical dysfunction in older recipient mice. Using single-cell transcriptomic analysis, we identified a naturally occurring senescent cell-like population in ADSCs primarily from old donors that resembles in vitro-generated senescent cells with regard to a number of key pathways. Our study reveals a previously unrecognized health concern due to ADSCs from old donors and lays the foundation for a new avenue of research to devise interventions to reduce harmful effects of ADSCs from old donors.

Producing Enhanced Yield and Nutritional Pigmentation in Lollo Rosso Through Manipulating the Irradiance, Duration, and Periodicity of LEDs in the Visible Region of Light.

Pigmented food are an important part of the human diet, and anthocyanins have demonstrable protection against tumor production in mouse models and beneficial effects on human liver chemistry. As such, producing pigmented crops is important for a nutritionally diverse diet. Lollo rosso lettuce is a fast-growing pigmented plant, is rich in phenolic compounds, and represents a suitable system to test optimization strategies for yield and anthocyanin production. High-energy UV wavebands are often used to stimulate increased pigmentation; however, we hypothesized that optimizing visible wavebands would deliver both yield and quality improvements. Growing Lollo rosso under irradiances between 5 and 180 W m-2 using visible waveband LEDs produced 0.4 g fresh weight per W m-2 in the linear portion of the curve between 5 and 40 W m-2 and achieved an approximate asymptote of 20 g fresh weight at around 100-120 W m-2 for yield. Anthocyanin content increased linearly with irradiance. We attempted to optimize the visible wavebands by supplementing half the asymptotic energy for 15 days with supplemental red (R) or blue (B) wavebands in the peaks of photosynthetic activity (430-460 and 630-660 nm). R and B affected rosette morphology with no significant impact on yield, but B significantly increased anthocyanin content by 94% compared to R. We therefore focused on further optimizing B by shortening the daily duration of supplemental B. The minimum B treatment that lacked significant pigment induction was 1 h. We hypothesized that short durations would be more active at different times in the diurnal cycle. Supplemental B was applied for 2 h at four different times. A night-break with B produced the highest yield and anthocyanin content. Our research demonstrates new ways to efficiently use readily available LEDs within the PAR wavebands to increase both yield and crop quality in controlled environment agriculture.

Single-cell analyses reveal increased intratumoral heterogeneity after the onset of therapy resistance in small-cell lung cancer.

The natural history of small cell lung cancer (SCLC) includes rapid evolution from chemosensitivity to chemoresistance, although mechanisms underlying this evolution remain obscure due to scarcity of post-relapse tissue samples. We generated circulating tumor cell (CTC)-derived xenografts (CDXs) from SCLC patients to study intratumoral heterogeneity (ITH) via single-cell RNAseq of chemo-sensitive and -resistant CDXs and patient CTCs. We found globally increased ITH including heterogeneous expression of therapeutic targets and potential resistance pathways, such as EMT, between cellular subpopulations following treatment-resistance. Similarly, serial profiling of patient CTCs directly from blood confirmed increased ITH post-relapse. These data suggest that treatment-resistance in SCLC is characterized by coexisting subpopulations of cells with heterogeneous gene expression leading to multiple, concurrent resistance mechanisms. These findings emphasize the need for clinical efforts to focus on rational combination therapies for treatment-naïve SCLC tumors to maximize initial responses and counteract the emergence of ITH and diverse resistance mechanisms.

Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population "antigen-presenting CAFs" and find that they activate CD4+ T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. SIGNIFICANCE: Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II-expressing CAFs with a capacity to present antigens to CD4+ T cells, and potentially to modulate the immune response in pancreatic tumors.See related commentary by Belle and DeNardo, p. 1001.This article is highlighted in the In This Issue feature, p. 983.

Single-Cell Transcriptome Analysis Reveals Estrogen Signaling Coordinately Augments One-Carbon, Polyamine, and Purine Synthesis in Breast Cancer.

Estrogen drives breast cancer (BCa) progression by directly activating estrogen receptor α (ERα). However, because of the stochastic nature of gene transcription, it is important to study the estrogen signaling pathway at the single-cell level to fully understand how ERα regulates transcription. Here, we performed single-cell transcriptome analysis on ERα-positive BCa cells following 17ß-estradiol stimulation and reconstructed the dynamic estrogen-responsive transcriptional network from discrete time points into a pseudotemporal continuum. Notably, differentially expressed genes show an estrogen-stimulated metabolic switch that favors biosynthesis but reduces estrogen degradation. Moreover, folate-mediated one-carbon metabolism is reprogrammed through the mitochondrial folate pathway and polyamine and purine synthesis are upregulated coordinately. Finally, we show AZIN1 and PPAT are direct ERα targets that are essential for BCa cell survival and growth. In summary, our study highlights the dynamic transcriptional heterogeneity in ERα-positive BCa cells upon estrogen stimulation and uncovers a mechanism of estrogen-mediated metabolic switch.

Author Correction: Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors.

In the version of the article published, the author list is not accurate. Igor Cima and Min-Han Tan should have been authors, appearing after Mark Wong in the author list, while Paul Jongjoon Choi should not have been listed as an author. Igor Cima and Min-Han Tan both have the affiliation Institute of Bioengineering and Nanotechnology, Singapore, Singapore, and their contributions should have been noted in the Author Contributions section as "I.C. preprocessed Primary Cell Atlas data with inputs from M.-H.T." The following description of the contribution of Paul Jongjoon Choi should not have appeared: "P.J.C. supported the smFISH experiments." In the 'RCA: global panel' section of the Online Methods, the following sentence should have appeared as the second sentence, "An expression atlas of human primary cells (the Primary Cell Atlas) was preprocessed similarly to in ref. 55," with new reference 55 (Cima, I. et al. Tumor-derived circulating endothelial cell clusters in colorectal cancer. Science Transl. Med. 8, 345ra89, 2016).

Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes.

Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. Thirty subtypes of RGCs have been identified to date. Here, we analyze 6225 RGCs (average of 5000 genes per cell) from right and left eyes by single-cell RNA-seq and classify them into 40 subtypes using clustering algorithms. We identify additional subtypes and markers, as well as transcription factors predicted to cooperate in specifying RGC subtypes. Zic1, a marker of the right eye-enriched subtype, is validated by immunostaining in situ. Runx1 and Fst, the markers of other subtypes, are validated in purified RGCs by fluorescent in situ hybridization (FISH) and immunostaining. We show the extent of gene expression variability needed for subtype segregation, and we show a hierarchy in diversification from a cell-type population to subtypes. Finally, we present a website for comparing the gene expression of RGC subtypes.