The latency associated with bone metastasis emergence in castrate‐resistant prostate cancer is attributed to dormancy, a state in which cancer cells persist prior to overt lesion formation. Using ...single‐cell transcriptomics and ex vivo profiling, we have uncovered the critical role of tumor‐intrinsic immune signaling in the retention of cancer cell dormancy. We demonstrate that loss of tumor‐intrinsic type I IFN occurs in proliferating prostate cancer cells in bone. This loss suppresses tumor immunogenicity and therapeutic response and promotes bone cell activation to drive cancer progression. Restoration of tumor‐intrinsic IFN signaling by HDAC inhibition increased tumor cell visibility, promoted long‐term antitumor immunity, and blocked cancer growth in bone. Key findings were validated in patients, including loss of tumor‐intrinsic IFN signaling and immunogenicity in bone metastases compared to primary tumors. Data herein provide a rationale as to why current immunotherapeutics fail in bone‐metastatic prostate cancer, and provide a new therapeutic strategy to overcome the inefficacy of immune‐based therapies in solid cancers.
Synopsis
Tumor‐intrinsic type I IFN is lost upon outgrowth of dormant prostate cancer cells in bone, driving metastasis. Therapeutic reversal of tumor‐intrinsic IFN loss enhances tumor cell visibility and the effectiveness of systemic immunomodulatory agents against bone‐metastasis.
Tumor‐intrinsic type I IFN and associated immune signaling is lost in prostate cancer cells that have spread to bone.
Tumor‐intrinsic type I IFN status is linked to the outgrowth of dormant prostate cancer cells in bone.
Therapeutic restoration of tumor‐intrinsic type I IFN inflates immunogenicity and immunotherapeutic response.
Tumor‐intrinsic type I IFN is lost upon outgrowth of dormant prostate cancer cells in bone, driving metastasis. Therapeutic reversal of tumor‐intrinsic IFN loss enhances tumor cell visibility and the effectiveness of systemic immunomodulatory agents against bone‐metastasis.
Changes in the cellular secretome are implicated in virus infection, malignancy, and anti-tumor immunity. We analyzed the association between transcriptional signatures (TS) from 24 different immune ...and stromal cell types on the prognosis of HPV-infected and HPV-free head and neck squamous carcinoma (HNSCC) patients from The Cancer Genome Atlas (TCGA) cohort. We found that HPV-positive HNSCC patients have tumors with elevated immune cell TS and improved prognosis, which was specifically associated with an increased tumor abundance of memory B and activated natural killer (NK) cell TS, compared to HPV-free HNSCC patients. HPV-infected patients upregulated many transcripts encoding secreted factors, such as growth factors, hormones, chemokines and cytokines, and their cognate receptors. Analysis of secretome transcripts and cognate receptors revealed that tumor expression of IL17RB and IL17REL are associated with a higher viral load and memory B and activated NK cell TS, as well as improved prognosis in HPV-infected HNSCC patients. The transcriptional parameters that we describe may be optimized to improve prognosis and risk stratification in the clinic and provide insights into gene and cellular targets that may potentially enhance anti-tumor immunity mediated by NK cells and memory B cells in HPV-infected HNSCC patients.
Glioblastoma (GBM) tumor cells exhibit drug resistance and are highly infiltrative. GBM stem cells (GSCs), which have low proliferative capacity are thought to be one of the sources of resistant ...cells which result in relapse/recurrence. However, the molecular mechanisms regulating quiescent-specific tumor cell biology are not well understood. Using human GBM cell lines and patient-derived GBM cells, Oregon Green dye retention was used to identify and isolate the slow-cycling, quiescent-like cell subpopulation from the more proliferative cells in culture. Sensitivity of cell subpopulations to temozolomide and radiation, as well as the migration and invasive potential were measured. Differential expression analysis following RNAseq identified genes enriched in the quiescent cell subpopulation. Orthotopic transplantation of cells into mice was used to compare the in vivo malignancy and invasive capacity of the cells. Proliferative quiescence correlated with better TMZ resistance and enhanced cell invasion, in vitro and in vivo. RNAseq expression analysis identified genes involved in the regulation cell invasion/migration and a three-gene signature, TGFBI, IGFBP3, CHI3L1, overexpressed in quiescent cells which correlates with poor GBM patient survival.
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•Determination of quiescence using a dye is superior to using GBM cell biomarkers.•Slow proliferating cells exhibit robust migration, invasion and drug resistance.•Slow proliferating cells overexpress mRNA of factors regulating invasion.•Overexpression of TGFBI, IGFBP3 and CHI3L1 correlates with poor GBM patient survival.
Evidence suggests that altered adipose tissue homeostasis may be an important contributor to the development and/or progression of prostate cancer. In this study, we investigated the adipose ...transcriptional profiles of low- and high-risk disease to determine both prognostic potential and possible biological drivers of aggressive disease. RNA was extracted from periprostatic adipose tissue from patients categorised as having prostate cancer with either a low or high risk of progression based on tumour characteristics at prostatectomy and profiled by RNA sequencing. The expression of selected genes was then quantified by qRT-PCR in a cross-validation cohort. In the first phase, a total of 677 differentially transcribed genes were identified, from which a subset of 14 genes was shortlisted. In the second phase, a 3 gene (IGHA1, OLFM4, RERGL) signature was refined and evaluated using recursive feature selection and cross-validation, obtaining a promising discriminatory utility (area under curve 0.72) at predicting the presence of high-risk disease. Genes implicated in immune and/or inflammatory responses predominated. Periprostatic adipose tissue from patients with high-risk prostate cancer has a distinct transcriptional signature that may be useful for detecting its occult presence. Differential expression appears to be driven by a local immune/inflammatory reaction to more advanced tumours, than any specific adipose tissue-specific tumour-promoting mechanism. This signature is transferable into a clinically usable PCR-based assay, which in a cross-validation cohort shows diagnostic potential.
The role of lymph node metastases in distant prostate cancer dissemination and lethality is ill defined. Patients with metastases restricted to lymph nodes have a better prognosis than those with ...distant metastatic spread, suggesting the possibility of distinct aetiologies. To explore this, we traced patterns of cancer dissemination using tumour phylogenies inferred from genome-wide copy-number profiling of 48 samples across 3 patients with lymph node metastatic disease and 3 patients with osseous metastatic disease. Our results show that metastatic cells in regional lymph nodes originate from evolutionary advanced extraprostatic tumour cells rather than less advanced central tumour cell populations. In contrast, osseous metastases do not exhibit such a constrained developmental lineage, arising from either intra or extraprostatic tumour cell populations, at early and late stages in the evolution of the primary. Collectively, this comparison suggests that lymph node metastases may not be an intermediate developmental step for distant osseous metastases, but rather represent a distinct metastatic lineage.
Obesity is linked with more aggressive prostate cancer and higher rates of disease recurrence post treatment. It is unclear if this is due to specific tumor-promoting effects of obesity or diagnostic ...bias. Patients undergoing prostatectomy were categorized according to their body mass index (BMI). Expected prostate-specific antigen (PSA) levels were calculated for each patient based on tumor characteristics. The effect of obesity on the accuracy of pre-treatment risk categorization was determined, and mediation analysis was used to identify the contribution of biologic vs non-biologic mechanisms to the observed increased risk of biochemical recurrence. Residual tumor-promoting effects were estimated in a survival model controlling for diagnostic error. The following results were obtained. The analysis included 1587 patients. Despite similar rates of adverse pathological features at prostatectomy, biochemical recurrence rates were significantly higher in very obese patients, which persisted after adjustment for stage, grade and PSA. Tumor volume however correlated significantly with BMI (P = 0.004), and the difference in predicted and observed ‘tumor-attributable’ PSA (Delta-PSA) in very obese patients was greater than three times higher than that of healthy patients (P = 0.0067). Regression analysis indicated that the effect of BMI on tumor volume was fully mediated indirectly by its effect on PSA. Inclusion of this diagnostic error as a covariate in the survival analysis attenuated the effect of BMI on recurrence. In conclusion, being very obese suppresses tumor-associated PSA resulting in a diagnostic bias that is responsible for errors in risk classification, and potentially contributes to a delay in initial presentation.
Abstract
Relative transcript abundance has proven to be a valuable tool for understanding the function of genes in biological systems. For the differential analysis of transcript abundance using RNA ...sequencing data, the negative binomial model is by far the most frequently adopted. However, common methods that are based on a negative binomial model are not robust to extreme outliers, which we found to be abundant in public datasets. So far, no rigorous and probabilistic methods for detection of outliers have been developed for RNA sequencing data, leaving the identification mostly to visual inspection. Recent advances in Bayesian computation allow large-scale comparison of observed data against its theoretical distribution given in a statistical model. Here we propose ppcseq, a key quality-control tool for identifying transcripts that include outlier data points in differential expression analysis, which do not follow a negative binomial distribution. Applying ppcseq to analyse several publicly available datasets using popular tools, we show that from 3 to 10 percent of differentially abundant transcripts across algorithms and datasets had statistics inflated by the presence of outliers.
Abstract
Malignant pleural effusions (MPE) are a common complication of advanced cancers, particularly those adjacent to the pleura such as lung and breast cancer and are a frequent complication in ...metastatic disease. The pathophysiology of MPE formation in advanced breast cancer remains poorly understood, and their composition and biology are understudied. To characterise the phenotypic diversity of malignant pleural effusion, we performed single-cell RNA sequencing on 10 MPEs from 7 metastatic breast cancer patients with diverse molecular subtypes: two triple negative (TNBC) patients, three luminal B patients including one with a rare inflammatory subtype, and two luminal A patients. For all patients, we sequenced cells from the entire MPE, without performing any enrichment or selection, in order to ascertain the cellular composition and molecular phenotypes in an unbiased manner. We identified pronounced differences in the relative proportions of malignant, mesothelial and immune cell populations: both TNBC and two Luminal B patients had extensive malignant cell populations, while the other three patients had few or no detectible malignant cells but extensive immune populations. We also observed heterogeneity in the expression of subtype-specific gene signatures and in copy number aberration patterns that captured variability across malignant cell populations both within and between patients. We observed that most malignant cells retained the molecular subtype diagnosed in the primary tumour, however sub-populations of malignant cells could be identified that mapped to different molecular subtypes, indicative of complexity in the molecular phenotypes of cancer cells disseminated to or metastasising in the pleural cavity. We distinguished mesothelial cell populations from malignant cells using key markers, expression of the PAM50 signatures, and copy number aberration patterns. We found that pleural mesothelial cells expressed a cancer associated fibroblast-like transcriptomic program that may support cancer growth within the pleural cavity through the secretion of growth factors that target malignant cells. Our dataset presents the first unbiased and unselected assessment of breast cancer associated MPEs at single cell resolution, providing the community with a vital resource for the study of MPEs. Our work highlights the molecular and cellular diversity captured in MPEs and advances the use of these clinically relevant biopsies both in monitoring disease progression and in the development of targeted therapeutics for patients with advanced breast cancer.
Citation Format: Holly J. Whitfield, Jean Berthelet, Stefano Mangiola, Caroline Bell, Robin L. Anderson, Bhupinder Pal, Anthony T. Papenfuss, Belinda Yeo, Delphine Merino, Melissa J. Davis. Defining the cellular composition and associated molecular phenotypes of malignant and non-malignant cells in breast cancer pleural effusions abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6232.
Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a
mutation. While this triple therapy has potent tumor-intrinsic ...effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic
melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103
dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses
While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.
Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical ...barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor-α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.
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