Truncated FGFR2 is a clinically actionable oncogene in multiple cancers.

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1-9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.

Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.

Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma.

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.

Cell-free DNA levels are increased in acute graft-versus-host disease.

BACKGROUND: Cell-free DNA (cfDNA) and nucleosomes, consisting of cfDNA and histones, are markers of cell activation and damage. In systemic inflammation these markers predict severity and fatality. However, the role of cfDNA in acute Graft-versus-Host Disease (aGvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), is unknown. OBJECTIVE: The aim of this study is to investigate the role of cfDNA as a marker of aGvHD. METHODS: We followed nucleosome levels in 37 allogeneic HSCT patients and an established xenotransplantation mouse model. We determined the origin of cfDNA with a species-specific polymerase chain reaction. RESULTS: In the plasma of aGvHD patients, nucleosome levels significantly increased around the time of aGvHD diagnosis compared to pretransplant, concurrently with a significant increase of known aGvHD markers ST2 and REG3α. In mice, we confirmed that nucleosomes were elevated during clinically detectable aGvHD. We found cfDNA to be mainly of human origin and to a lesser extent of mouse origin, indicating that cfDNA is released by (proliferating) human xeno-reactive PBMC and damaged mouse cells. CONCLUSION: We show increased cfDNA both in an aGvHD mouse model and in aGvHD patients. We also demonstrate that donor hematopoietic cells and to a lesser degree (damaged) host cells are the cellular source of cfDNA in aGvHD. We propose that nucleosomes and cfDNA might be an additive marker for aGvHD.

Exogenous ERα Expression in the Mammary Epithelium Decreases Over Time and Does Not Contribute to p53-Deficient Mammary Tumor Formation in Mice.

Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.

BRCA1-associated mammary tumorigenesis is dependent on estrogen rather than progesterone signaling.

Hereditary breast cancers in BRCA1 mutation carriers are mostly estrogen receptor α (ERα)-negative and progesterone receptor (PR)-negative; however, hormone depletion via bilateral oophorectomy does result in a marked reduction in breast cancer risk, suggesting that BRCA1-associated breast tumorigenesis is dependent on hormone signaling. We used geneticaly engineered mouse models to determine the individual influences of ERα and PR signaling on the development of BRCA1-deficient breast cancer. In line with the human data, BRCA1-deficient mouse mammary tumors are ERα-negative, and bilateral ovariectomy leads to abrogation of mammary tumor development. Hormonal replacement experiments in ovariectomized mice showed that BRCA1-deficient mammary tumor formation is promoted by estrogen but not by progesterone. In line with these data, mammary tumorigenesis was significantly delayed by the selective ERα downregulator fulvestrant, but not by the selective PR antagonist Org33628. Together, our results illustrate that BRCA1-associated tumorigenesis is dependent on estrogen signaling rather than on progesterone signaling, and call into question the utility of PR antagonists as a tumor prevention strategy for BRCA1 mutation carriers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

XenofilteR: computational deconvolution of mouse and human reads in tumor xenograft sequence data.

BACKGROUND: Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary. RESULTS: We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations. CONCLUSIONS: XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR .

Prophylactic window therapy with the clinical poly(ADP-ribose) polymerase inhibitor olaparib delays BRCA1-deficient mammary tumour formation in mice.

Women with heterozygous germline mutations in the BRCA1 tumour suppressor gene are strongly predisposed to developing early-onset breast cancer through loss of the remaining wild-type BRCA1 allele and inactivation of TP53. Although tumour prevention strategies in BRCA1-mutation carriers are still limited to prophylactic surgery, several therapeutic strategies have been developed to target the DNA repair defects (also known as 'BRCAness') of BRCA1-deficient tumours. In particular, DNA-damaging agents such as platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors show strong activity against BRCA1-mutated tumours. However, it is unclear whether drugs that target BRCAness can also be used to prevent tumour formation in BRCA1-mutation carriers, especially as loss of wild-type BRCA1 may not be the first event in BRCA1-associated tumourigenesis. We performed prophylactic treatments in a genetically engineered mouse model in which de novo development of BRCA1-deficient mammary tumours is induced by stochastic loss of BRCA1 and p53. We found that prophylactic window therapy with nimustine, cisplatin or olaparib reduced the amount and size of mammary gland lesions, and significantly increased the median tumour latency. Similar results were obtained with intermittent prophylactic treatment with olaparib. Importantly, prophylactic window therapy with nimustine and cisplatin resulted in an increased fraction of BRCA1-proficient mammary tumours, suggesting selective survival and malignant transformation of BRCA1-proficient lesions upon prophylactic treatment with DNA-damaging agents. Prophylactic therapy with olaparib significantly prolonged mammary tumour-free survival without any significant increase in the fraction of BRCA1-proficient tumours, warranting the evaluation of this PARP inhibitor in prophylactic trials in BRCA1-mutation carriers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

GATA1-Deficient Dendritic Cells Display Impaired CCL21-Dependent Migration toward Lymph Nodes Due to Reduced Levels of Polysialic Acid.

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KODC), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KODC mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KODC DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.

p120-Catenin Is Critical for the Development of Invasive Lobular Carcinoma in Mice.

Loss of E-cadherin expression is causal to the development of invasive lobular breast carcinoma (ILC). E-cadherin loss leads to dismantling of the adherens junction and subsequent translocation of p120-catenin (p120) to the cytosol and nucleus. Although p120 is critical for the metastatic potential of ILC through the regulation of Rock-dependent anoikis resistance, it remains unknown whether p120 also contributes to ILC development. Using genetically engineered mouse models with mammary gland-specific inactivation of E-cadherin, p120 and p53, we demonstrate that ILC formation induced by E-cadherin and p53 loss is severely impaired upon concomitant inactivation of p120. Tumors that developed in the triple-knockout mice were mostly basal sarcomatoid carcinomas that displayed overt nuclear atypia and multinucleation. In line with the strong reduction in ILC incidence in triple-knockout mice compared to E-cadherin and p53 double-knockout mice, no functional redundancy of p120 family members was observed in mouse ILC development, as expression and localization of ARVCF, p0071 or δ-catenin was unaltered in ILCs from triple-knockout mice. In conclusion, we show that loss of p120 in the context of the p53-deficient mouse models is dominant over E-cadherin inactivation and its inactivation promotes the development of basal, epithelial-to-mesenchymal-transition (EMT)-type invasive mammary tumors.

LGR6-dependent conditional inactivation of E-cadherin and p53 leads to invasive skin and mammary carcinomas in mice.

Tissue-specific inactivation of E-cadherin combined with tumor suppressor loss leads to invasive and metastatic cancers in mice. While epidermal E-cadherin loss in mice induces squamous cell carcinomas, inactivation of E-cadherin in the mammary gland leads to invasive lobular carcinoma. To further explore the carcinogenic consequences of cell-cell adhesion loss in these compartments, we developed a new conditional mouse model inactivating E-cadherin (Cdh1) and p53 (Trp53) simultaneously in cells expressing the leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6), a putative epithelial stem cell marker in the skin and alveolar progenitor marker in the mammary gland. Compound Lgr6-CreERT2;Cdh1F;Trp53F female mice containing either heterozygous or homozygous Cdh1F alleles were bred, and Lgr6-driven Cre expression was activated in pre-puberal mice using tamoxifen. We observed that 41% of the mice (16/39) developed mostly invasive squamous-type skin carcinomas, but also a non-lobular mammary tumor was formed. In contrast to previous K14cre or WAPcre E-cadherin and p53 compound models, no significant differences were detected in the tumor-free survival of Lgr6-CreERT2 heterozygous Cdh1F/WT;Trp53F/F versus homozygous Cdh1F/F;Trp53F/F mice (778 versus 754 days, p=0.5). One Cdh1F homozygous mouse presented with lung metastasis that originated from a non-lobular and ERα negative invasive mammary gland carcinoma with squamous metaplasia. In total, 2/8 (25%) Cdh1F heterozygous and 3/12 (25%) Cdh1F homozygous mice developed metastases to lungs, liver, lymph nodes, or the gastro-intestinal tract. In conclusion, we show that inducible and conditional Lgr6-driven inactivation of E-cadherin and p53 in mice causes squamous cell carcinomas of the skin in approximately 40% of the mice and an occasional ductal-type mammary carcinoma after long latency periods.

Carboxylesterase 1 family knockout alters drug disposition and lipid metabolism.

The mammalian carboxylesterase 1 (Ces1/CES1) family comprises several enzymes that hydrolyze many xenobiotic chemicals and endogenous lipids. To investigate the pharmacological and physiological roles of Ces1/CES1, we generated Ces1 cluster knockout (Ces1 -/- ) mice, and a hepatic human CES1 transgenic model in the Ces1 -/- background (TgCES1). Ces1 -/- mice displayed profoundly decreased conversion of the anticancer prodrug irinotecan to SN-38 in plasma and tissues. TgCES1 mice exhibited enhanced metabolism of irinotecan to SN-38 in liver and kidney. Ces1 and hCES1 activity increased irinotecan toxicity, likely by enhancing the formation of pharmacodynamically active SN-38. Ces1 -/- mice also showed markedly increased capecitabine plasma exposure, which was moderately decreased in TgCES1 mice. Ces1 -/- mice were overweight with increased adipose tissue, white adipose tissue inflammation (in males), a higher lipid load in brown adipose tissue, and impaired blood glucose tolerance (in males). These phenotypes were mostly reversed in TgCES1 mice. TgCES1 mice displayed increased triglyceride secretion from liver to plasma, together with higher triglyceride levels in the male liver. These results indicate that the carboxylesterase 1 family plays essential roles in drug and lipid metabolism and detoxification. Ces1 -/- and TgCES1 mice will provide excellent tools for further study of the in vivo functions of Ces1/CES1 enzymes.

Natural deletion of mouse carboxylesterases Ces1c/d/e impacts drug metabolism and metabolic syndrome development.

Mammalian carboxylesterase 1 enzymes can hydrolyze many xenobiotic chemicals and endogenous lipids. We here identified and characterized a mouse strain (FVB/NKI) in which three of the eight Ces1 genes were spontaneously deleted, removing Ces1c and Ces1e partly, and Ces1d entirely. We studied the impact of this Ces1c/d/e deficiency on drug and lipid metabolism and homeostasis. Ces1c/d/e-/- mice showed strongly impaired conversion of the anticancer prodrug irinotecan to its active metabolite SN-38 in plasma, spleen and lung. Plasma hydrolysis of the oral anticancer prodrug capecitabine to 5-DFCR was also profoundly reduced in Ces1c/d/e-/- mice. Our findings resolved previously unexplained FVB/NKI pharmacokinetic anomalies. On a medium-fat diet, Ces1c/d/e-/- female mice exhibited moderately higher body weight, mild inflammation in gonadal white adipose tissue (gWAT), and increased lipid load in brown adipose tissue (BAT). Ces1c/d/e-/- males showed more pronounced inflammation in gWAT and an increased lipid load in BAT. On a 5-week high-fat diet exposure, Ces1c/d/e deficiency predisposed to developing obesity, enlarged and fatty liver, glucose intolerance and insulin resistance, with severe inflammation in gWAT and increased lipid load in BAT. Hepatic proteomics analysis revealed that the acute phase response, involved in the dynamic cycle of immunometabolism, was activated in these Ces1c/d/e-/- mice. This may contribute to the obesity-related chronic inflammation and adverse metabolic disease in this strain. While Ces1c/d/e deficiency clearly exacerbated metabolic syndrome development, long-term (18-week) high-fat diet exposure overwhelmed many, albeit not all, observed phenotypic differences.

Neoadjuvant immune checkpoint blockade triggers persistent and systemic Treg activation which blunts therapeutic efficacy against metastatic spread of breast tumors.

The clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response.

CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer.

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice, we show that CAFs in both invasive lobular breast cancer and triple-negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo and in vitro studies reveal the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. Functional co-culture experiments show that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data suggest that CD26+ and CD26- NFs transform into distinct CAF subpopulations in mouse models of breast cancer.

MYC is a clinically significant driver of mTOR inhibitor resistance in breast cancer.

Targeting the PI3K-AKT-mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K-AKT-mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies.

Pro-mutagenic effects of the gut microbiota in a Lynch syndrome mouse model.

The gut microbiota strongly impacts the development of sporadic colorectal cancer (CRC), but it is largely unknown how the microbiota affects the pathogenesis of mismatch-repair-deficient CRC in the context of Lynch syndrome. In a mouse model for Lynch syndrome, we found a nearly complete loss of intestinal tumor development when animals were transferred from a conventional "open" animal facility to specific-pathogen-free (SPF) conditions. Using 16S sequencing we detected large changes in microbiota composition between the two facilities. Transcriptomic analyses of tumor-free intestinal tissues showed signs of strong intestinal inflammation in conventional mice. Whole exome sequencing of tumors developing in Msh2-Lynch mice revealed a much lower mutational load in the single SPF tumor than in tumors developing in conventional mice, suggesting reduced epithelial proliferation in SPF mice. Fecal microbiota transplantations with conventional feces altered the immune landscape and gut homeostasis, illustrated by increased gut length and elevated epithelial proliferation and migration. This was associated with drastic changes in microbiota composition, in particular increased relative abundances of different mucus-degrading taxa such as Desulfovibrio and Akkermansia, and increased bacterial-epithelial contact. Strikingly, transplantation of conventional microbiota increased microsatellite instability in untransformed intestinal epithelium of Msh2-Lynch mice, indicating that the composition of the microbiota influences the rate of mutagenesis in MSH2-deficient crypts.

RNF31 inhibition sensitizes tumors to bystander killing by innate and adaptive immune cells.

Tumor escape mechanisms for immunotherapy include deficiencies in antigen presentation, diminishing adaptive CD8+ T cell antitumor activity. Although innate natural killer (NK) cells are triggered by loss of MHC class I, their response is often inadequate. To increase tumor susceptibility to both innate and adaptive immune elimination, we performed parallel genome-wide CRISPR-Cas9 knockout screens under NK and CD8+ T cell pressure. We identify all components, RNF31, RBCK1, and SHARPIN, of the linear ubiquitination chain assembly complex (LUBAC). Genetic and pharmacologic ablation of RNF31, an E3 ubiquitin ligase, strongly sensitizes cancer cells to NK and CD8+ T cell killing. This occurs in a tumor necrosis factor (TNF)-dependent manner, causing loss of A20 and non-canonical IKK complexes from TNF receptor complex I. A small-molecule RNF31 inhibitor sensitizes colon carcinoma organoids to TNF and greatly enhances bystander killing of MHC antigen-deficient tumor cells. These results merit exploration of RNF31 inhibition as a clinical pharmacological opportunity for immunotherapy-refractory cancers.

Multiparametric Analyses of Hepatocellular Carcinoma Somatic Mouse Models and Their Associated Tumor Microenvironment.

The rising incidence and increasing mortality of hepatocellular carcinoma (HCC), combined with its high tumor heterogeneity, lack of druggable targets, and tendency to develop resistance to chemotherapeutics, make the development of better models for this cancer an urgent challenge. To better mimic the high diversity within the HCC genetic landscape, versatile somatic murine models have recently been developed using the hydrodynamic tail vein injection (HDTVi) system. These represent novel in vivo tools to interrogate HCC phenotype and response to therapy, and importantly, allow further analyses of the associated tumor microenvironment (TME) shaped by distinct genetic backgrounds. Here, we describe several optimized protocols to generate, collect, and experimentally utilize various samples obtained from HCC somatic mouse models generated by HDTVi. More specifically, we focus on techniques relevant to ex vivo analyses of the complex liver TME using multiparameter flow cytometric analyses of over 21 markers, immunohistochemistry, immunofluorescence, and histochemistry. We describe the transcriptional assessment of whole tissue, or of isolated immune subsets by flow-cytometry-based cell sorting, and other protein-oriented analyses. Together, these streamlined protocols allow the optimal use of each HCC murine model of interest and will assist researchers in deciphering the relations between cancer cell genetics and systemic and local changes in immune cell landscapes in the context of HCC progression. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of HCC mouse models by hydrodynamic tail vein injection Basic Protocol 2: Assessment of HCC tumor progression by magnetic resonance imaging Basic Protocol 3: Mouse sacrifice and sample collection in HCC mouse models Support Protocol 1: Preparation of serum or plasma from blood Basic Protocol 4: Single-cell preparation and HCC immune landscape phenotyping by flow cytometry Alternate Protocol 1: Flow cytometric analysis of circulating immune cells Support Protocol 2: Generation, maintenance, and characterization of HCC cell lines Support Protocol 3: Fluorescence-activated cell sorting of liver single-cell preparation Basic Protocol 5: Preparation and immunohistochemical analysis of tumor tissues from HCC-bearing liver Alternate Protocol 2: Preparation and analyses for immunofluorescence staining of HCC-bearing liver Support Protocol 4: Liver-specific phenotypic analyses of liver sections Support Protocol 5: Immunohistochemical quantification in liver sections Basic Protocol 6: Preparation of snap-frozen tumor tissue from extracted liver and transcriptional analyses of bulk tumor or sorted cells Alternate Protocol 3: Protein analyses from HCC samples and serum or plasma.