Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses.

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain T cell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ T cell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ T cell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

Serpins promote cancer cell survival and vascular co-option in brain metastasis.

Brain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for the initiation of brain metastasis in lung and breast cancers.

Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability.

Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies.

Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer.

The complex interactions between tumors and their microenvironment remain to be elucidated. Combining large-scale approaches, we examined the spatio-temporal dynamics of 28 different immune cell types (immunome) infiltrating tumors. We found that the immune infiltrate composition changed at each tumor stage and that particular cells had a major impact on survival. Densities of T follicular helper (Tfh) cells and innate cells increased, whereas most T cell densities decreased along with tumor progression. The number of B cells, which are key players in the core immune network and are associated with prolonged survival, increased at a late stage and showed a dual effect on recurrence and tumor progression. The immune control relevance was demonstrated in three endoscopic orthotopic colon-cancer mouse models. Genomic instability of the chemokine CXCL13 was a mechanism associated with Tfh and B cell infiltration. CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis correlating with survival. This integrative study reveals the immune landscape in human colorectal cancer and the major hallmarks of the microenvironment associated with tumor progression and recurrence.

Metastatic colonization by circulating tumour cells.

Metastasis is the main cause of death in people with cancer. To colonize distant organs, circulating tumour cells must overcome many obstacles through mechanisms that we are only now starting to understand. These include infiltrating distant tissue, evading immune defences, adapting to supportive niches, surviving as latent tumour-initiating seeds and eventually breaking out to replace the host tissue. They make metastasis a highly inefficient process. However, once metastases have been established, current treatments frequently fail to provide durable responses. An improved understanding of the mechanistic determinants of such colonization is needed to better prevent and treat metastatic cancer.

Therapy-induced tumour secretomes promote resistance and tumour progression.

Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer.

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Cancer Evolution: A Multifaceted Affair.

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery. SIGNIFICANCE: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.

Mutations in GNA11 in uveal melanoma.

BACKGROUND: Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas. METHODS: We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi. RESULTS: We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway. CONCLUSIONS: Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.).

Disruption of Trp53 in livers of mice induces formation of carcinomas with bilineal differentiation.

BACKGROUND & AIMS: p53 limits the self-renewal of stem cells from various tissues. Loss of p53, in combination with other oncogenic events, results in aberrant self-renewal and transformation of progenitor cells. It is not known whether loss of p53 is sufficient to induce tumor formation in liver. METHODS: We used AlfpCre mice to create mice with liver-specific disruption of Trp53 (AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice). We analyzed colony formation and genomic features and gene expression patterns in liver cells during hepatocarcinogenesis in mice with homozygous, heterozygous, and no disruption of Trp53. RESULTS: Liver-specific disruption of Trp53 consistently induced formation of liver carcinomas that had bilineal differentiation. In nontransformed liver cells and cultured primary liver cells, loss of p53 (but not p21) resulted in chromosomal imbalances and increased clonogenic capacity of liver progenitor cells (LPCs) and hepatocytes. Primary cultures of hepatocytes and LPCs from AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice, but not Cdkn1a(-/-) mice, formed tumors with bilineal differentiation when transplanted into immunocompromised mice. Spontaneous liver tumors that developed in AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice had significant but complex alterations in expression of Rb checkpoint genes compared with chemically induced liver tumors that developed mice with wild-type Trp53. CONCLUSIONS: Deletion of p53 from livers of mice is sufficient to induce tumor formation. The tumors have bilineal differentiation and dysregulation of Rb checkpoint genes.

Human Papillomavirus 42 Drives Digital Papillary Adenocarcinoma and Elicits a Germ Cell-like Program Conserved in HPV-Positive Cancers.

The skin is exposed to viral pathogens, but whether they contribute to the oncogenesis of skin cancers has not been systematically explored. Here we investigated 19 skin tumor types by analyzing off-target reads from commonly available next-generation sequencing data for viral pathogens. We identified human papillomavirus 42 (HPV42) in 96% (n = 45/47) of digital papillary adenocarcinoma (DPA), an aggressive cancer occurring on the fingers and toes. We show that HPV42, so far considered a nononcogenic, "low-risk" HPV, recapitulates the molecular hallmarks of oncogenic, "high-risk" HPVs. Using machine learning, we find that HPV-driven transformation elicits a germ cell-like transcriptional program conserved throughout all HPV-driven cancers (DPA, cervical carcinoma, and head and neck cancer). We further show that this germ cell-like transcriptional program, even when reduced to the top two genes (CDKN2A and SYCP2), serves as a fingerprint of oncogenic HPVs with implications for early detection, diagnosis, and therapy of all HPV-driven cancers. SIGNIFICANCE: We identify HPV42 as a uniform driver of DPA and add a new member to the short list of tumorigenic viruses in humans. We discover that all oncogenic HPVs evoke a germ cell-like transcriptional program with important implications for detecting, diagnosing, and treating all HPV-driven cancers. See related commentary by Starrett et al., p. 17. This article is highlighted in the In This Issue feature, p. 1.

High-resolution analyses of copy number changes in disseminated tumor cells of patients with breast cancer.

The presence of disseminated tumor cells (DTCs) in bone marrow (BM) identifies breast cancer patients with less favorable outcome. Furthermore, molecular characterization is required to investigate the malignant potential of these cells. This study presents a single-cell array comparative genomic hybridization (SCaCGH) method providing molecular analysis of immunomorphologically detected DTCs. The resolution limit of the method was estimated using the cancer cell line SK-BR-3 on 44 and 244k arrays. The technique was further tested on 28 circulating tumor cells and four hematopoietic cells (HCs) from peripheral blood (n = 8 patients). The SCaCGH method was finally applied to 24 DTCs, three immunopositive cells morphologically classified as probable HCs from breast cancer patients and five HC controls from BM (n = 7 patients plus n = 1 healthy donor). The frequency of copy number changes of the DTCs revealed similarities with primary breast tumor samples. Three of the patients had available profiles for DTCs and the corresponding tumor tissue from primary surgery. More than two-third of the analyzed DTCs disclosed equivalent changes, both to each other and to the corresponding primary disease, whereas the rest of the cells showed balanced profiles. The probable HCs revealed either balanced profiles (n = 2) or changes comparable to the tumor tissue and DTCs (n = 1), indicating morphological overlap between HCs and DTCs. Similar aberration patterns were visible in DTCs collected at diagnosis and at 3 years relapse-free follow-up. SCaCGH may be a powerful tool for the molecular characterization of DTCs.

Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS).

Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells.

Mechanism-based combination therapies for metastatic cancer.

Functional interrogation of cancer and immune cells during tumor evolution reveals cancer drivers and informs mechanism-based combination therapies.

Defining 'chromosomal instability'.

Most scientists agree that the majority of human solid malignant tumors are characterized by chromosomal instability (CIN) involving gain or loss of whole chromosomes or fractions of chromosomes. CIN is thought to be an early event during tumorigenesis and might therefore be involved in tumor initiation. Despite its frequent occurrence in tumors and its potential importance in tumor evolution, CIN is poorly defined and is used inconsistently and imprecisely. Here, we provide criteria to define CIN and argue that few experimental approaches are capable of assessing the presence of CIN. Accurate assessment of CIN is crucial to elucidate whether CIN is a driving force for tumorigenesis and whether a chromosomally unstable genome is necessary for tumor progression.

Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum-free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays.

Clinical DNA is often available in limited quantities requiring whole-genome amplification for subsequent genome-wide assessment of copy-number variation (CNV) by array-CGH. In pre-implantation diagnosis and analysis of micrometastases, even merely single cells are available for analysis. However, procedures allowing high-resolution analyses of CNVs from single cells well below resolution limits of conventional cytogenetics are lacking. Here, we applied amplification products of single cells and of cell pools (5 or 10 cells) from patients with developmental delay, cancer cell lines and polar bodies to various oligo tiling array platforms with a median probe spacing as high as 65 bp. Our high-resolution analyses reveal that the low amounts of template DNA do not result in a completely unbiased whole genome amplification but that stochastic amplification artifacts, which become more obvious on array platforms with tiling path resolution, cause significant noise. We implemented a new evaluation algorithm specifically for the identification of small gains and losses in such very noisy ratio profiles. Our data suggest that when assessed with sufficiently sensitive methods high-resolution oligo-arrays allow a reliable identification of CNVs as small as 500 kb in cell pools (5 or 10 cells), and of 2.6-3.0 Mb in single cells.

Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters.

We developed a functional lineage tracing tool termed CaTCH (CRISPRa tracing of clones in heterogeneous cell populations). CaTCH combines precise clonal tracing of millions of cells with the ability to retrospectively isolate founding clones alive before and during selection, allowing functional experiments. Using CaTCH, we captured rare clones representing as little as 0.001% of a population and investigated the emergence of resistance to targeted melanoma therapy in vivo.

Down-regulation of A20 promotes immune escape of lung adenocarcinomas.

Inflammation is a well-known driver of lung tumorigenesis. One strategy by which tumor cells escape tight homeostatic control is by decreasing the expression of the potent anti-inflammatory protein tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20. We observed that tumor cell intrinsic loss of A20 markedly enhanced lung tumorigenesis and was associated with reduced CD8+ T cell-mediated immune surveillance in patients with lung cancer and in mouse models. In mice, we observed that this effect was completely dependent on increased cellular sensitivity to interferon-γ (IFN-γ) signaling by aberrant activation of TANK-binding kinase 1 (TBK1) and increased downstream expression and activation of signal transducer and activator of transcription 1 (STAT1). Interrupting this autocrine feed forward loop by knocking out IFN-α/ß receptor completely restored infiltration of cytotoxic T cells and rescued loss of A20 depending tumorigenesis. Downstream of STAT1, programmed death ligand 1 (PD-L1) was highly expressed in A20 knockout lung tumors. Accordingly, immune checkpoint blockade (ICB) treatment was highly efficient in mice harboring A20-deficient lung tumors. Furthermore, an A20 loss-of-function gene expression signature positively correlated with survival of melanoma patients treated with anti-programmed cell death protein 1. Together, we have identified A20 as a master immune checkpoint regulating the TBK1-STAT1-PD-L1 axis that may be exploited to improve ICB therapy in patients with lung adenocarcinoma.

Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumor microenvironment and cross-resistance to immunotherapy in melanoma.

How targeted therapies and immunotherapies shape tumors, and thereby influence subsequent therapeutic responses, is poorly understood. In the present study, we show, in melanoma patients and mouse models, that when tumors relapse after targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies. We find that cross-resistance is mediated by a cancer cell-instructed, immunosuppressive tumor microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response. Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumors to immunotherapy. Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which not only is reactivated, but also exhibits an increased transcriptional output that drives immune evasion. Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.