Single-cell transcriptomics reveals opposing roles of Shp2 in Myc-driven liver tumor cells and microenvironment

Cell reports

2021 Nov 09

Chen, WS;Liang, Y;Zong, M;Liu, JJ;Kaneko, K;Hanley, KL;Zhang, K;Feng, GS;
PMID: 34758313 | DOI: 10.1016/j.celrep.2021.109974

The mechanisms of Myc-driven liver tumorigenesis are inadequately understood. Herein we show that Myc-driven hepatocellular carcinoma (HCC) is dramatically aggravated in mice with hepatocyte-specific Ptpn11/Shp2 deletion. However, Myc-induced tumors develop selectively from the rare Shp2-positive hepatocytes in Shp2-deficent liver, and Myc-driven oncogenesis depends on an intact Ras-Erk signaling promoted by Shp2 to sustain Myc stability. Despite a stringent requirement of Shp2 cell autonomously, Shp2 deletion induces an immunosuppressive environment, resulting in defective clearance of tumor-initiating cells and aggressive tumor progression. The basal Wnt/β-catenin signaling is upregulated in Shp2-deficient liver, which is further augmented by Myc transfection. Ablating Ctnnb1 suppresses Myc-induced HCC in Shp2-deficient livers, revealing an essential role of β-catenin. Consistently, Myc overexpression and CTNNB1 mutations are frequently co-detected in HCC patients with poor prognosis. These data elucidate complex mechanisms of liver tumorigenesis driven by cell-intrinsic oncogenic signaling in cooperation with a tumor-promoting microenvironment generated by disrupting the specific oncogenic pathway.

A Chemokine Regulatory Loop Induces Cholesterol Synthesis in Lung-Colonizing Triple-Negative Breast Cancer Cells to Fuel Metastatic Growth

Molecular therapy : the journal of the American Society of Gene Therapy

2021 Jul 15

Han, B;Alonso-Valenteen, F;Wang, Z;Deng, N;Lee, TY;Gao, B;Zhang, Y;Xu, Y;Zhang, X;Billet, S;Fan, X;Shiao, S;Bhowmick, N;Medina-Kauwe, L;Giuliano, A;Cui, X;
PMID: 34274535 | DOI: 10.1016/j.ymthe.2021.07.003

Triple-negative breast cancer (TNBC) has a high propensity for organ-specific metastasis. However, the underlying mechanisms are not well understood. Here, we show that the primary TNBC tumor-derived C-X-C motif chemokines 1/2/8 (CXCL1/2/8) stimulate lung resident fibroblasts to produce C-C motif chemokines 2/7 (CCL2/7), which in turn activate cholesterol synthesis in lung-colonizing TNBC cells and induce angiogenesis at lung metastatic sites. Inhibiting cholesterol synthesis in lung-colonizing breast tumor cells by the pulmonary administration of simvastatin-carrying HER3-targeting nanoparticles reduces the angiogenesis and growth of lung metastases in a syngeneic TNBC mouse model. Our findings reveal a novel, chemokine-regulated mechanism for the cholesterol synthesis pathway and a critical role of metastatic site-specific cholesterol synthesis in the pulmonary tropism of TNBC metastasis. The study has implications for the unresolved epidemiological observation that the use of cholesterol-lowering drugs has no effect on breast cancer incidence but can unexpectedly reduce breast cancer mortality, suggesting interventions of cholesterol synthesis in lung metastases as an effective treatment to improve survival in TNBC patients.

Microglia drive transient insult-induced brain injury by chemotactic recruitment of CD8+ T lymphocytes

Neuron

2022 Dec 29

Shi, Z;Yu, P;Lin, WJ;Chen, S;Hu, X;Chen, S;Cheng, J;Liu, Q;Yang, Y;Li, S;Zhang, Z;Xie, J;Jiang, J;He, B;Li, Y;Li, H;Xu, Y;Zeng, J;Huang, J;Mei, J;Cai, J;Chen, J;Wu, LJ;Ko, H;Tang, Y;
PMID: 36603584 | DOI: 10.1016/j.neuron.2022.12.009

The crosstalk between the nervous and immune systems has gained increasing attention for its emerging role in neurological diseases. Radiation-induced brain injury (RIBI) remains the most common medical complication of cranial radiotherapy, and its pathological mechanisms have yet to be elucidated. Here, using single-cell RNA and T cell receptor sequencing, we found infiltration and clonal expansion of CD8+ T lymphocytes in the lesioned brain tissues of RIBI patients. Furthermore, by strategies of genetic or pharmacologic interruption, we identified a chemotactic action of microglia-derived CCL2/CCL8 chemokines in mediating the infiltration of CCR2+/CCR5+ CD8+ T cells and tissue damage in RIBI mice. Such a chemotactic axis also participated in the progression of cerebral infarction in the mouse model of ischemic injury. Our findings therefore highlight the critical role of microglia in mediating the dysregulation of adaptive immune responses and reveal a potential therapeutic strategy for non-infectious brain diseases.

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

Mod Pathol.

2016 Sep 30

Son SM, Ha SY, Yoo HY, Oh D, Kim SJ, Kim WS, Ko YH.
PMID: 27687005 | DOI: 10.1038/modpathol.2016.56

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

TGFβ inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence

Sci Transl Med.

2018 Aug 15

Bird TG, Müller M, Boulter L, Vincent DF, Ridgway RA, Lopez-Guadamillas E, Lu WY, Jamieson T, Govaere O, Campbell AD, Ferreira-Gonzalez S, Cole AM, Hay T, Simpson KJ, Clark W, Hedley A, Clarke M, Gentaz P, Nixon C, Bryce S, Kiourtis C, Sprangers J, Nibbs
PMID: 30111642 | DOI: 10.1126/scitranslmed.aan1230

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-β1 (TGFβ1) ligand. In acetaminophen poisoning, inhibition of TGFβ receptor 1 (TGFβR1) improved mouse survival. TGFβR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

A single-cell transcriptome atlas of glial diversity in the human hippocampus across the postnatal lifespan

Cell stem cell

2022 Nov 03

Su, Y;Zhou, Y;Bennett, ML;Li, S;Carceles-Cordon, M;Lu, L;Huh, S;Jimenez-Cyrus, D;Kennedy, BC;Kessler, SK;Viaene, AN;Helbig, I;Gu, X;Kleinman, JE;Hyde, TM;Weinberger, DR;Nauen, DW;Song, H;Ming, GL;
PMID: 36332572 | DOI: 10.1016/j.stem.2022.09.010

The molecular diversity of glia in the human hippocampus and their temporal dynamics over the lifespan remain largely unknown. Here, we performed single-nucleus RNA sequencing to generate a transcriptome atlas of the human hippocampus across the postnatal lifespan. Detailed analyses of astrocytes, oligodendrocyte lineages, and microglia identified subpopulations with distinct molecular signatures and revealed their association with specific physiological functions, age-dependent changes in abundance, and disease relevance. We further characterized spatiotemporal heterogeneity of GFAP-enriched astrocyte subpopulations in the hippocampal formation using immunohistology. Leveraging glial subpopulation classifications as a reference map, we revealed the diversity of glia differentiated from human pluripotent stem cells and identified dysregulated genes and pathological processes in specific glial subpopulations in Alzheimer's disease (AD). Together, our study significantly extends our understanding of human glial diversity, population dynamics across the postnatal lifespan, and dysregulation in AD and provides a reference atlas for stem-cell-based glial differentiation.

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy

Science immunology

2022 Apr 01

Hoch, T;Schulz, D;Eling, N;Gómez, JM;Levesque, MP;Bodenmiller, B;
PMID: 35363540 | DOI: 10.1126/sciimmunol.abk1692

Intratumoral immune cells are crucial for tumor control and antitumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by binding of specific immune cell receptors to chemokines, a class of secreted chemotactic cytokines. To broadly characterize chemokine expression and function in melanoma, we used multiplexed mass cytometry-based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most of the profiled chemokines and exhibited low levels of antigen presentation and markers of inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing the B lymphocyte chemoattractant CXCL13. In tumors with B cells but no B cell follicles, T cells were the sole source of CXCL13, suggesting that T cells play a role in B cell recruitment and potentially in B cell follicle formation. B cell patches and follicles were also enriched with TCF7+ naïve-like T cells, a cell type that is predictive of response to immune checkpoint blockade. Our data highlight the strength of targeted RNA and protein codetection to analyze tumor immune microenvironments based on chemokine expression and suggest that the formation of tertiary lymphoid structures may be accompanied by naïve and naïve-like T cell recruitment, which may contribute to antitumor activity.

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping

Cell

2018 Aug 30

Di Liberto G, Pantelyushin S, Kreutzfeldt M, Page N, Musardo S, Coras R, Steinbach K, Vincenti I, Klimek B, Lingner T, Salinas G, Lin-Marq N, Staszewski O, Costa Jordão MJ, Wagner I, Egervari K, Mack M, Bellone C, Blümcke I, Prinz M, Pinschewer DD, Merkle
PMID: - | DOI: 10.1016/j.cell.2018.07.049

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen’s encephalitis patients. In this devastating CD8+T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

PDGFRβ Cells Rapidly Relay Inflammatory Signal from the Circulatory System to Neurons via Chemokine CCL2

Neuron

2018 Sep 27

Duan L, Zhang XD, Miao WX, Sun YJ, Xiong G, Wu Q, Li G, Yang P, Yu H, Li H, Wang Y, Zhang M, Hu LY, Tong X, Zhou WH, Yu X.
PMID: - | DOI: 10.1016/j.neuron.2018.08.030

Acute infection, if not kept in check, can lead to systemic inflammatory responses in the brain. Here, we show that within 2 hr of systemic inflammation, PDGFRβ mural cells of blood vessels rapidly secrete chemokine CCL2, which in turn increases total neuronal excitabilityby promoting excitatory synaptic transmission in glutamatergic neurons of multiple brain regions. By single-cell RNA sequencing, we identified Col1a1 and Rgs5 subgroups of PDGFRβ cells as the main source of CCL2. Lipopolysaccharide (LPS)- or Poly(I:C)-treated pericyte culture medium induced similar effects in a CCL2-dependent manner. Importantly, in Pdgfrb-Cre;Ccl2fl/fl mice, LPS-induced increase in excitatory synaptic transmission was significantly attenuated. These results demonstrate in vivo that PDGFRβ cells function as initial sensors of external insults by secreting CCL2, which relays the signal to the central nervous system. Through their gateway position in the brain, PDGFRβ cells are ideally positioned to respond rapidly to environmental changes and to coordinate responses.

Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration

The EMBO journal

2021 May 05

Guillermin, O;Angelis, N;Sidor, CM;Ridgway, R;Baulies, A;Kucharska, A;Antas, P;Rose, MR;Cordero, J;Sansom, O;Li, VSW;Thompson, BJ;
PMID: 33950519 | DOI: 10.15252/embj.2020105770

Wnt signalling induces a gradient of stem/progenitor cell proliferation along the crypt-villus axis of the intestine, which becomes expanded during intestinal regeneration or tumour formation. The YAP transcriptional co-activator is known to be required for intestinal regeneration, but its mode of regulation remains controversial. Here we show that the YAP-TEAD transcription factor is a key downstream effector of Wnt signalling in the intestine. Loss of YAP activity by Yap/Taz conditional knockout results in sensitivity of crypt stem cells to apoptosis and reduced cell proliferation during regeneration. Gain of YAP activity by Lats1/2 conditional knockout is sufficient to drive a crypt hyperproliferation response. In particular, Wnt signalling acts transcriptionally to induce YAP and TEAD1/2/4 expression. YAP normally localises to the nucleus only in crypt base stem cells, but becomes nuclear in most intestinal epithelial cells during intestinal regeneration after irradiation, or during organoid growth, in a Src family kinase-dependent manner. YAP-driven crypt expansion during regeneration involves an elongation and flattening of the Wnt signalling gradient. Thus, Wnt and Src-YAP signals cooperate to drive intestinal regeneration.

Rapid Loss of RNA Detection by In Situ Hybridization in Stored Tissue Blocks and Preservation by Cold Storage of Unstained Slides

American Journal of Clinical Pathology

2017 Oct 09

Baena-Del Valle JA, Zheng Q, Hicks JL, Trock HFBJ, Morrissey C, Corey E, Cornish TC, Sfanos KS, De Marzo AM.
PMID: - | DOI: 10.1093/ajcp/aqx094

Abstract

Objectives

Recent commercialization of methods for in situ hybridization using Z-pair probe/branched DNA amplification has led to increasing adoption of this technology for interrogating RNA expression in formalin-fixed, paraffin-embedded (FFPE) tissues. Current practice for FFPE block storage is to maintain them at room temperature, often for many years.

Methods

To examine the effects of block storage time on FFPE tissues using a number of RNA in situ probes with the Advanced Cellular Diagnostic’s RNAscope assay.

Results

We report marked reductions in signals after 5 years and significant reductions often after 1 year. Furthermore, storing unstained slides cut from recent cases (<1 year old) at –20°C can preserve hybridization signals significantly better than storing the blocks at room temperature and cutting the slides fresh when needed.

Conclusions

We submit that the standard practice of storing FFPE tissue blocks at room temperature should be reevaluated to better preserve RNA for in situ hybridization.

A novel role for C–C motif chemokine receptor 2 during infection with hypervirulent Mycobacterium tuberculosis

Mucosal Immunol.

2018 Aug 16

Dunlap MD, Howard N, Das S, Scott N, Ahmed M, Prince O, Rangel-Moreno J, Rosa BA, Martin J, Kaushal D, Kaplan G, Mitreva M, Kim KW, Randolph GJ, Khader SA.
PMID: 30115997 | DOI: 10.1038/s41385-018-0071-y

C-C motif chemokine receptor 2 (CCR2) is a major chemokine axis that recruits myeloid cells including monocytes and macrophages. Thus far, CCR2-/- mice have not been found to be susceptible to infection with Mycobacterium tuberculosis (Mtb). Here, using a prototype W-Beijing family lineage 2 Mtb strain, HN878, we show that CCR2-/- mice exhibit increased susceptibility to tuberculosis (TB). Following exposure to Mtb HN878, alveolar macrophages (AMs) are amongst the earliest cells infected. We show that AMs accumulate early in the airways following infection and express CCR2. During disease progression, CCR2-expressing AMs exit the airways and localize within the TB granulomas. RNA-sequencing of sorted airway and non-airway AMs from infected mice show distinct gene expression profiles, suggesting that upon exit from airways and localization within granulomas, AMs become classically activated. The absence of CCR2+ cells specifically at the time of AM egress from the airways resulted in enhanced susceptibility to Mtb infection. Furthermore, infection with an Mtb HN878 mutant lacking phenolic glycolipid (PGL) expression still resulted in increased susceptibility in CCR2-/- mice. Together, these data show a novel rolefor CCR2 in protective immunity against clinically relevant Mtb infections.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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