Spatial proximity of tumor-immune interactions predicts patient outcome in hepatocellular carcinoma.

BACKGROUND AND AIMS: The fitness and viability of a tumor ecosystem are influenced by the spatial organization of its cells. We aimed to study the structure, architecture, and cell-cell dynamics of the heterogeneous liver cancer tumor microenvironment using spatially resolved multiplexed imaging. APPROACH AND RESULTS: We performed co-detection by indexing multiplexed immunofluorescence imaging on 68 HCC biopsies from Thai patients [(Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC)] as a discovery cohort, and then validated the results in an additional 190 HCC biopsies from Chinese patients [Liver Cancer Institute (LCI)]. We segmented and annotated 117,270 and 465,632 cells from the TIGER-LC and LCI cohorts, respectively. We observed 4 patient groups of TIGER-LC (IC1, IC2, IC3, and IC4) with distinct tumor-immune cellular interaction patterns. In addition, patients from IC2 and IC4 had much better overall survival than those from IC1 and IC3. Noticeably, tumor and CD8 + T-cell interactions were strongly enriched in IC2, the group with the best patient outcomes. The close proximity between the tumor and CD8 + T cells was a strong predictor of patient outcome in both the TIGER-LC and the LCI cohorts. Bulk transcriptomic data from 51 of the 68 HCC cases were used to determine tumor-specific gene expression features of our classified subtypes. Moreover, we observed that the presence of immune spatial neighborhoods in HCC as a measure of overall immune infiltration is linked to better patient prognosis. CONCLUSIONS: Highly multiplexed imaging analysis of liver cancer reveals tumor-immune cellular heterogeneity within spatial contexts, such as tumor and CD8 + T-cell interactions, which may predict patient survival.

Single-cell biology uncovers apoptotic cell death and its spatial organization as a potential modifier of tumor diversity in HCC.

BACKGROUND AND AIMS: HCC is a highly aggressive and heterogeneous cancer type with limited treatment options. Identifying drivers of tumor heterogeneity may lead to better therapeutic options and favorable patient outcomes. We investigated whether apoptotic cell death and its spatial architecture is linked to tumor molecular heterogeneity using single-cell in situ hybridization analysis. APPROACH AND RESULTS: We analyzed 254 tumor samples from two HCC cohorts using tissue microarrays. We developed a mathematical model to quantify cellular diversity among HCC samples using two tumor markers, cyclin-dependent kinase inhibitor 3 and protein regulator of cytokinesis 1 as surrogates for heterogeneity and caspase 3 (CASP3) as an apoptotic cell death marker. We further explored the impact of potential dying-cell hubs on tumor cell diversity and patient outcome by density contour mapping and spatial proximity analysis. We also developed a selectively controlled in vitro model of cell death using CRISPR/CRISPR-associated 9 to determine therapy response and growth under hypoxic conditions. We found that increasing levels of CASP3+ tumor cells are associated with higher tumor diversity. Interestingly, we discovered regions of densely populated CASP3+ , which we refer to as CASP3+ cell islands, in which the nearby cellular heterogeneity was found to be the greatest compared to cells farther away from these islands and that this phenomenon was associated with survival. Additionally, cell culture experiments revealed that higher levels of cell death, accompanied by increased CASP3 expression, led to greater therapy resistance and growth under hypoxia. CONCLUSIONS: These results are consistent with the hypothesis that increased apoptotic cell death may lead to greater tumor heterogeneity and thus worse patient outcomes.

Toward a Liver Cell Atlas: Understanding Liver Biology in Health and Disease at Single-Cell Resolution.

Single-cell technologies are revolutionizing our understanding of cellular heterogeneity and functional diversity in health and disease. Here, we review the current knowledge and advances in liver biology using single-cell approaches. We focus on the landscape of the composition and the function of cells in a healthy liver in the context of its spatial organization. We also highlight the alterations of the molecular landscape in chronic liver disease and liver cancer, which includes the identification of disease-related cell types, altered cellular functions, dynamic cell-cell interactions, the plasticity of malignant cells, the collective behavior of a cell community, and microenvironmental reprogramming. We anticipate that the uncovered liver cell atlas will help deciphering the molecular and cellular mechanisms driving a healthy liver into a disease state. It also offers insight into the detection of new therapeutic targets and paves the way for effective disease interventions.

Single-cell atlas of tumor cell evolution in response to therapy in hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intratumor molecular heterogeneity is a key feature of tumorigenesis and is linked to treatment failure and patient prognosis. Herein, we aimed to determine what drives tumor cell evolution by performing single-cell transcriptomic analysis. METHODS: We analyzed 46 hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) biopsies from 37 patients enrolled in interventional studies at the NIH Clinical Center, with 16 biopsies collected before and after treatment from 7 patients. We developed a novel machine learning-based consensus clustering approach to track cellular states of 57,000 malignant and non-malignant cells including tumor cell transcriptome-based functional clonality analysis. We determined tumor cell relationships using RNA velocity and reverse graph embedding. We also studied longitudinal samples from 4 patients to determine tumor cellular state and its evolution. We validated our findings in bulk transcriptomic data from 488 patients with HCC and 277 patients with iCCA. RESULTS: Using transcriptomic clusters as a surrogate for functional clonality, we observed an increase in tumor cell state heterogeneity which was tightly linked to patient prognosis. Furthermore, increased functional clonality was accompanied by a polarized immune cell landscape which included an increase in pre-exhausted T cells. We found that SPP1 expression was tightly associated with tumor cell evolution and microenvironmental reprogramming. Finally, we developed a user-friendly online interface as a knowledge base for a single-cell atlas of liver cancer. CONCLUSIONS: Our study offers insight into the collective behavior of tumor cell communities in liver cancer as well as potential drivers of tumor evolution in response to therapy. LAY SUMMARY: Intratumor molecular heterogeneity is a key feature of tumorigenesis that is linked to treatment failure and patient prognosis. In this study, we present a single-cell atlas of liver tumors from patients treated with immunotherapy and describe intratumoral cell states and their hierarchical relationship. We suggest osteopontin, encoded by the gene SPP1, as a candidate regulator of tumor evolution in response to treatment.

Estrogen-related receptor ß activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.

Glioblastoma (GBM; grade 4 glioma) is a highly aggressive and incurable tumor. GBM has recently been characterized as highly dependent on alternative splicing, a critical driver of tumor heterogeneity and plasticity. Estrogen-related receptor ß (ERR-ß) is an orphan nuclear receptor expressed in the brain, where alternative splicing of the 3' end of the pre-mRNA leads to the production of 3 validated ERR-ß protein products: ERR-ß short form (ERR-ßsf), ERR-ß2, and ERR-ß exon 10 deleted. Our prior studies have shown the ERR-ß2 isoform to play a role in G2/M cell cycle arrest and induction of apoptosis, in contrast to the function of the shorter ERR-ßsf isoform in senescence and G1 cell cycle arrest. In this study, we sought to better define the role of the proapoptotic ERR-ß2 isoform in GBM. We show that the ERR-ß2 isoform is located not only in the nucleus but also in the cytoplasm. ERR-ß2 suppresses GBM cell migration and interacts with the actin nucleation-promoting factor cortactin, and an ERR-ß agonist is able to remodel the actin cytoskeleton and similarly suppress GBM cell migration. We further show that inhibition of the splicing regulatory cdc2-like kinases in combination with an ERR-ß agonist shifts isoform expression in favor of ERR-ß2 and potentiates inhibition of growth and migration in GBM cells and intracranial tumors.-Tiek, D. M., Khatib, S. A., Trepicchio, C. J., Heckler, M. M., Divekar, S. D., Sarkaria, J. N., Glasgow, E., Riggins, R. B. Estrogen-related receptor ß activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.

Resveratrol inhibits obesity-associated adipose tissue dysfunction and tumor growth in a mouse model of postmenopausal claudin-low breast cancer.

Adipose tissue dysregulation, a hallmark of obesity, contributes to a chronic state of low-grade inflammation and is associated with increased risk and progression of several breast cancer subtypes, including claudin-low breast tumors. Unfortunately, mechanistic targets for breaking the links between obesity-associated adipose tissue dysfunction, inflammation, and claudin-low breast cancer growth have not been elucidated. Ovariectomized female C57BL/6 mice were randomized (n = 15/group) to receive a control diet, a diet-induced obesity (DIO) diet, or a DIO + resveratrol (0.5% wt/wt) diet. Mice consumed these diets ad libitum throughout study and after 6 weeks were orthotopically injected with M-Wnt murine mammary tumor cells, a model of estrogen receptor (ER)-negative claudin-low breast cancer. Compared with controls, DIO mice displayed adipose dysregulation and metabolic perturbations including increased mammary adipocyte size, cyclooxygenase-2 (COX-2) expression, inflammatory eicosanoid levels, macrophage infiltration, and prevalence of crown-like structures (CLS). DIO mice (relative to controls) also had increased systemic inflammatory cytokines and decreased adipocyte expression of peroxisome proliferator-activated receptor gamma (PPARγ) and other adipogenesis-regulating genes. Supplementing the DIO diet with resveratrol prevented obesity-associated increases in mammary tumor growth, mammary adipocyte hypertrophy, COX-2 expression, macrophage infiltration, CLS prevalence, and serum cytokines. Resveratrol also offset the obesity-associated downregulation of adipocyte PPARγ and other adipogenesis genes in DIO mice. Our findings suggest that resveratrol may inhibit obesity-associated inflammation and claudin-low breast cancer growth by inhibiting adipocyte hypertrophy and associated adipose tissue dysregulation that typically accompanies obesity.

Reducing the burden of obesity-associated cancers with anti-inflammatory long-chain omega-3 polyunsaturated fatty acids.

Today's world population has an unprecedented risk of dying from the consequences of being overweight and obese. Chronic diseases such as cardiovascular disease, type 2 diabetes, and cancer are often accelerated because of excessive adiposity. Various biological mechanisms are implicated in the obesity-cancer link, particularly local and systemic inflammation as well as altered growth factor signaling pathways. In order to combat obesity-induced inflammation and the resulting increases in cancer risk and progression, the identification of safe and effective mechanism-based interventions is imperative. Notably, long chain omega-3 polyunsaturated fatty acids (PUFAs) modulate the secretion of pro-inflammatory cytokines, prostaglandins and other inflammatory mediators, restore insulin sensitivity, and can prevent or delay tumorigenesis. Delineating the precise mechanisms by which omega-3 PUFAs suppress obesity-induced inflammation will help identify promising key mechanistic targets and intervention strategies to break the obesity-cancer link.

Calorie restriction outperforms bariatric surgery in a murine model of obesity and triple-negative breast cancer.

Obesity promotes triple-negative breast cancer (TNBC), and effective interventions are urgently needed to break the obesity-TNBC link. Epidemiologic studies indicate that bariatric surgery reduces TNBC risk, while evidence is limited or conflicted for weight loss via low-fat diet (LFD) or calorie restriction (CR). Using a murine model of obesity-driven TNBC, we compared the antitumor effects of vertical sleeve gastrectomy (VSG) with LFD, chronic CR, and intermittent CR. Each intervention generated weight and fat loss and suppressed tumor growth relative to obese mice (greatest suppression with CR). VSG and CR regimens exerted both similar and unique effects, as assessed using multiomics approaches, in reversing obesity-associated transcript, epigenetics, secretome, and microbiota changes and restoring antitumor immunity. Thus, in a murine model of TNBC, bariatric surgery and CR each reverse obesity-driven tumor growth via shared and distinct antitumor mechanisms, and CR is superior to VSG in reversing obesity's procancer effects.

Causes and functional intricacies of inter- and intratumor heterogeneity of primary liver cancers.

Tumor heterogeneity is a major feature of primary liver cancers. Defined as the unique genotypic and phenotypic differences of cancer cells within a single tumor (intratumor) or amongst different patients (intertumor), tumor heterogeneity has consistently been linked to worse clinical outcomes in most, if not all, solid tumor types. In particular, liver cancer heterogeneity has been associated with altered immune infiltration, resistance to therapeutics, and worse overall patient survival. Current advancements in single-cell omic technologies have allowed for a deeper understanding and appreciation of the intricate composition and relationships between individual cells within a tumor. These observations have led to the discovery of new cell types in liver cancer, potential new mechanisms of therapy resistance and tumor progression, and new insights into the evolutionary patterns of liver cancer. To better understand the tumor biology of liver cancers and their heterogeneous features, we will begin this chapter on a brief background of liver cancer and then discuss the various etiologies of this disease and how each one can contribute to diverse genomic, transcriptomic, proteomic, and spatial architecture observations. Next, we will go into the specific causes and implications of tumor heterogeneity and end with how understanding the spatial architecture of liver tumors can provide us with new insights and ideas for tumor diversity and therapeutic development.

Multiregional single-cell dissection of tumor and immune cells reveals stable lock-and-key features in liver cancer.

Intratumor heterogeneity may result from the evolution of tumor cells and their continuous interactions with the tumor microenvironment which collectively drives tumorigenesis. However, an appearance of cellular and molecular heterogeneity creates a challenge to define molecular features linked to tumor malignancy. Here we perform multiregional single-cell RNA sequencing (scRNA-seq) analysis of seven liver cancer patients (four hepatocellular carcinoma, HCC and three intrahepatic cholangiocarcinoma, iCCA). We identify cellular dynamics of malignant cells and their communication networks with tumor-associated immune cells, which are validated using additional scRNA-seq data of 25 HCC and 12 iCCA patients as a stable fingerprint embedded in a malignant ecosystem representing features of tumor aggressiveness. We further validate the top ligand-receptor interaction pairs (i.e., LGALS9-SLC1A5 and SPP1-PTGER4 between tumor cells and macrophages) associated with unique transcriptome in additional 542 HCC patients. Our study unveils stable molecular networks of malignant ecosystems, which may open a path for therapeutic exploration.

Understanding the Cause and Consequence of Tumor Heterogeneity.

Tumor heterogeneity is a large conundrum in cancer medicine, making most therapeutic interventions palliative rather than curative. Here we discuss the implications of how molecularly targeted therapies in solid malignancies that promote limited cancer cell death may in fact make tumors more heterogeneous, increase aggressive phenotypes, and thus worsen patient outcomes.

PKM2 inhibition may reverse therapeutic resistance to transarterial chemoembolization in hepatocellular carcinoma.

BACKGROUND: Therapeutic options for patients with hepatocellular carcinoma (HCC) are limited. Transarterial chemoembolization (TACE) is an interventional procedure used to deliver chemotherapy and embolizing agents directly to the tumor and is the procedure of choice for patients with intermediate stage HCC. While effective, more than 40% of patients do not respond to therapy, highlighting the need to investigate possible mechanisms of resistance. We sought to evaluate mechanisms of TACE resistance and evaluate a potential therapeutic target to overcome this resistance. METHODS: Using a prognostic gene signature which predicts TACE response (TACE Navigator) in a cohort of HCC patients who received TACE, patients were classified as responders and non-responders. Transcriptomic and gene pathway analysis were used to identify potential drivers of TACE resistance. Knockdown of the gene encoding rate limiting enzyme PKM2 using shRNA in HCC cell lines, as well as pharmacologic inhibition of PKM2 with shikonin using an in vitro TACE model measured response to chemotherapy under hypoxia. Finally, we replicated the TACE model with shikonin using patient derived cell line organoids (PDC). Functional studies were performed in vitro using immunoblotting, quantitative polymerase chain reaction, glycolysis and hypoxia assays. RESULTS: In patient non-responders, we identified enrichment of the glycolysis pathway, specifically of the gene encoding the rate-limiting enzyme PKM2. We identified four HCC cell lines which recapitulated a TACE responder-like and non-responder-like phenotype. PKM2 knockdown in HCC cell lines demonstrated a less proliferative and aggressive phenotype as well as improved drug sensitivity to both doxorubicin and cisplatin. In vitro TACE model demonstrated that TACE non-responder-like cells overcame therapeutic resistance and rendered them susceptible to therapy through PKM2 knockdown. Lastly, we obtained similar results using a pharmacologic PKM2 inhibitor, shikonin in both cell lines, and PDC organoids. CONCLUSION: Elevated PKM2 is associated with treatment resistance and abbreviated survival in patients receiving TACE. Elevated PKM2 in vitro is associated with increased utilization of the glycolysis pathway, resulting in oxygen independent cell metabolism. Through PKM2 knockdown as well as with pharmacologic inhibition with shikonin, non-responder cells can be reprogrammed to act as responders and could improve TACE efficacy in patients.

AGO2 Mediates MYC mRNA Stability in Hepatocellular Carcinoma.

Deregulated RNA-binding proteins (RBP), such as Argonaute 2 (AGO2), mediate tumor-promoting transcriptomic changes during carcinogenesis, including hepatocellular carcinoma (HCC). While AGO2 is well characterized as a member of the RNA-induced silencing complex (RISC), which represses gene expression through miRNAs, its role as a bona fide RBP remains unclear. In this study, we investigated AGO2's role as an RBP that regulates the MYC transcript to promote HCC. Using mRNA and miRNA arrays from patients with HCC, we demonstrate that HCCs with elevated AGO2 levels are more likely to have the mRNA transcriptome deregulated and are associated with poor survival. Moreover, AGO2 overexpression stabilizes the MYC transcript independent of miRNAs. These observations provide a novel mechanism of gene regulation by AGO2 and provide further insights into the potential functions of AGO2 as an RBP in addition to RISC. IMPLICATIONS: Authors demonstrate that the RBP Argonaute 2 stabilizes the MYC transcript to promote HCC.

Genome-Wide RNAi Screen Identifies PMPCB as a Therapeutic Vulnerability in EpCAM+ Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a genetically heterogeneous disease for which a dominant actionable molecular driver has not been identified. Patients with the stem cell-like EpCAM+AFP+ HCC subtype have poor prognosis. Here, we performed a genome-wide RNAi screen to identify genes with a synthetic lethal interaction with EpCAM as a potential therapeutic target for the EpCAM+AFP+ HCC subtype. We identified 26 candidate genes linked to EpCAM/Wnt/ß-catenin signaling and HCC cell growth. We further characterized the top candidate PMPCB, which plays a role in mitochondrial protein processing, as a bona fide target for EpCAM+ HCC. PMPCB blockage suppressed EpCAM expression and Wnt/ß-catenin signaling via mitochondria-related reactive oxygen species production and FOXO activities, resulting in apoptosis and tumor suppression. These results indicate that a synthetic lethality screen is a viable strategy to identify actionable drivers of HCC and identify PMPCB as a therapeutically vulnerable gene in EpCAM+ HCC subpopulations. SIGNIFICANCE: This study identifies PMPCB as critical to mitochondrial homeostasis and a synthetic lethal candidate that selectively kills highly resistant EpCAM+ HCC tumors by inactivating the Wnt/ß-catenin signaling pathway.

Leptin Signaling Mediates Obesity-Associated CSC Enrichment and EMT in Preclinical TNBC Models.

Obesity is associated with poor prognosis in triple-negative breast cancer (TNBC). Preclinical models of TNBC were used to test the hypothesis that increased leptin signaling drives obesity-associated TNBC development by promoting cancer stem cell (CSC) enrichment and/or epithelial-to-mesenchymal transition (EMT). MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like, triple-negative mammary tumors, received either a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat) for up to 42 weeks (n = 15/group). Mice were monitored for tumor development and euthanized when tumor diameter reached 1.5 cm. Tumoral gene expression was assessed via RNA sequencing (RNA-seq). DIO mice had greater body weight and percent body fat at termination than controls. DIO mice, versus controls, demonstrated reduced survival, increased systemic metabolic and inflammatory perturbations, upregulated tumoral CSC/EMT gene signature, elevated tumoral aldehyde dehydrogenase activity (a CSC marker), and greater leptin signaling. In cell culture experiments using TNBC cells (murine: E-Wnt and M-Wnt; human: MDA-MB-231), leptin enhanced mammosphere formation, and media supplemented with serum from DIO versus control mice increased cell viability, migration, invasion, and CSC- and EMT-related gene expression, including Foxc2, Twist2, Vim, Akt3, and Sox2 In E-Wnt cells, knockdown of leptin receptor ablated these procancer effects induced by DIO mouse serum. These findings indicate that increased leptin signaling is causally linked to obesity-associated TNBC development by promoting CSC enrichment and EMT.Implications: Leptin-associated signals impacting CSC and EMT may provide new targets and intervention strategies for decreasing TNBC burden in obese women. Mol Cancer Res; 16(5); 869-79. ©2018 AACR.

Energy Balance Modulation Impacts Epigenetic Reprogramming, ERα and ERß Expression, and Mammary Tumor Development in MMTV-neu Transgenic Mice.

The association between obesity and breast cancer risk and prognosis is well established in estrogen receptor (ER)-positive disease but less clear in HER2-positive disease. Here, we report preclinical evidence suggesting weight maintenance through calorie restriction (CR) may limit risk of HER2-positive breast cancer. In female MMTV-HER2/neu transgenic mice, we found that ERα and ERß expression, mammary tumorigenesis, and survival are energy balance dependent in association with epigenetic reprogramming. Mice were randomized to receive a CR, overweight-inducing, or diet-induced obesity regimen (n = 27/group). Subsets of mice (n = 4/group/time point) were euthanized after 1, 3, and 5 months to characterize diet-dependent metabolic, transcriptional, and epigenetic perturbations. Remaining mice were followed up to 22 months. Relative to the overweight and diet-induced obesity regimens, CR decreased body weight, adiposity, and serum metabolic hormones as expected and also elicited an increase in mammary ERα and ERß expression. Increased DNA methylation accompanied this pattern, particularly at CpG dinucleotides located within binding or flanking regions for the transcriptional regulator CCCTC-binding factor of ESR1 and ESR2, consistent with sustained transcriptional activation of ERα and ERß. Mammary expression of the DNA methylation enzyme DNMT1 was stable in CR mice but increased over time in overweight and diet-induced obesity mice, suggesting CR obviates epigenetic alterations concurrent with chronic excess energy intake. In the survival study, CR elicited a significant suppression in spontaneous mammary tumorigenesis. Overall, our findings suggest a mechanistic rationale to prevent or reverse excess body weight as a strategy to reduce HER2-positive breast cancer risk. Cancer Res; 77(9); 2500-11. ©2017 AACR.

Obesity-Associated Alterations in Inflammation, Epigenetics, and Mammary Tumor Growth Persist in Formerly Obese Mice.

Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable with control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL6 levels, expression of proinflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus nonobese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression. Cancer Prev Res; 9(5); 339-48. ©2016 AACR.