Targeting senescent cells in translational medicine.

Organismal ageing is a complex process driving progressive impairment of functionality and regenerative potential of tissues. Cellular senescence is a state of stable cell cycle arrest occurring in response to damage and stress and is considered a hallmark of ageing. Senescent cells accumulate in multiple organs during ageing, contribute to tissue dysfunction and give rise to pathological manifestations. Senescence is therefore a defining feature of a variety of human age-related disorders, including cancer, and targeted elimination of these cells has recently emerged as a promising therapeutic approach to ameliorate tissue damage and promote repair and regeneration. In addition, in vivo identification of senescent cells has significant potential for early diagnosis of multiple pathologies. Here, we review existing senolytics, small molecules and drug delivery tools used in preclinical therapeutic strategies involving cellular senescence, as well as probes to trace senescent cells. We also review the clinical research landscape in senescence and discuss how identifying and targeting cellular senescence might positively affect pathological and ageing processes

A Two-Photon Probe Based on Naphthalimide-Styrene Fluorophore for the In Vivo Tracking of Cellular Senescence

Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal β-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues

Development of Patient Derived Xenograft Models of Overt Spontaneous Breast Cancer Metastasis: A Cautionary Note.

Several approaches are being evaluated to improve the historically limited value of studying transplanted primary tumors derived by injection of cells from established cell lines for predicting subsequent cancer therapy outcomes in patients and clinical trials. These approaches include use of genetically engineered mouse models (GEMMs) of spontaneous tumors, or patient tumor tissue derived xenografts (PDXs). Almost all such therapy studies utilizing such models involve treatment of established primary tumors. An alternative approach we have developed involves transplanted human tumor xenografts derived from established cell lines to treat mice with overt visceral metastases after primary tumor resection. The rationale is to mimic the more challenging circumstance of treating patients with late stage metastatic disease. These metastatic models entail prior in vivo selection of heritable, phenotypically stable variants with increased aggressiveness for spontaneous metastasis; they were derived by orthotopic injection of tumor cells followed by primary tumor resection and serial selection of distant spontaneous metastases, from which variant cell lines having a more aggressive heritable metastatic phenotype were established. We attempted to adopt this strategy for breast cancer PDXs. We studied five breast cancer PDXs, with the emphasis on two, called HCI-001 and HCI-002, both derived from triple negative breast cancer patients. However significant technical obstacles were encountered. These include the inherent slow growth rates of PDXs, the rarity of overt spontaneous metastases (detected in only 3 of 144 mice), very high rates of tumor regrowths at the primary tumor resection site, the failure of the few human PDX metastases isolated to manifest a more aggressive metastatic phenotype upon re-transplantation into new hosts, and the formation of metastases which were derived from de novo mouse thymomas arising in aged SCID mice that we used for the experiments. We discuss several possible strategies that may be employed to overcome these limitations. Uncovering the basis of the failure to detect a high rate of overt spontaneous distant metastases having a heritable phenotype in PDX models may reveal new insights into the biology and treatment of advanced metastatic disease

Acceleration of tumor growth of the variants derived from a metastasis.

<p>Graph showing the tumor growth rate of consecutive passages of two different tumors and their variants derived from lung metastases and representative slides stained for H&E: <b>A.</b> HCI-001; <b>B.</b> HCI-001 LM1; <b>C.</b> HCI-002; and <b>D.</b> HCI-002 LM2. Tumors did not show an increase in their growth rate with successive passages. The variants derived from lung metastases have an accelerated growth when implanted orthotopically as primary tumors in the mfp compared to the parental tumors. Scale bars, 150 μm.</p

<i>In vivo</i> selection of a PDX breast cancer-derived metastatic variant.

<p><b>A.</b> SCID mice were implanted in the mammary fat pad (mfp) with tumor fragments of the triple negative breast cancer PDX tumor HCI-002. <b>B.</b> Two months later, primary tumors were resected. <b>C.</b> After 7 months, one mouse developed on overt lung metastasis that was positive for the Human Leukocyte Antigen (HLA). <b>D.</b> Fragments of this lung metastasis were implanted in the mammary fat pads (mfp) of naive SCID mice and the variant called HCI-002 ML2 was isolated. This new variant grew with an accelerated rate when compared to the parental HCI-002 PDX. <b>E.</b> HCI-002 LM2 tumors were resected and mice kept alive. <b>F.</b> Seven months after primary tumor resection a spontaneous lung metastases was detected. <b>G.</b> Pieces of the metastatic nodule found in the lungs were implanted in the mfp of naive SCID mice, creating a new variant, called HCI-002 LM2-1.</p

A versatile drug delivery system targeting senescent cells.

Senescent cells accumulate in multiple aging-associated diseases, and eliminating these cells has recently emerged as a promising therapeutic approach. Here, we take advantage of the high lysosomal β-galactosidase activity of senescent cells to design a drug delivery system based on the encapsulation of drugs with galacto-oligosaccharides. We show that gal-encapsulated fluorophores are preferentially released within senescent cells in mice. In a model of chemotherapy-induced senescence, gal-encapsulated cytotoxic drugs target senescent tumor cells and improve tumor xenograft regression in combination with palbociclib. Moreover, in a model of pulmonary fibrosis in mice, gal-encapsulated cytotoxics target senescent cells, reducing collagen deposition and restoring pulmonary function. Finally, gal-encapsulation reduces the toxic side effects of the cytotoxic drugs. Drug delivery into senescent cells opens new diagnostic and therapeutic applications for senescence-associated disorders

Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.We are grateful to Tom Nightingale and Maria Niklison-Chirou for reading the manuscript. Alissa Weaver provided tagged CD63 constructs; and Jacob Yount and I-Chueh Huang supplied the IFITM3 and shIFITM3 plasmids. We are grateful to Luke Gammon, the Queen Mary University of London (QMUL) Genome Centre, and Gary Warnes for excellent technical support. Mouse hepatic stellate cells were a gift from Scott Lowe. A.O.’s lab is supported by the BBSRC (BB/P000223/1) and The Royal Society(RG170399). M.B. is funded by the MRC (MR/K501372/1) and the Centre for Genomics and Child Health. P.C.-F. (IN606B 2017/014) and J.F.-L.(ED481B 2017/117) are funded by the Xunta de Galicia.S

The NALCN channel regulates metastasis and nonmalignant cell dissemination.

Funder: Cancer Research UK (CRUK); doi: https://doi.org/10.13039/501100000289Funder: American Lebanese Syrian Associated Charities (ALSAC); doi: https://doi.org/10.13039/100012524Funder: U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics); doi: https://doi.org/10.13039/100011541Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI); doi: https://doi.org/10.13039/100000054Funder: RCUK | MRC | Medical Research Foundation; doi: https://doi.org/10.13039/501100009187We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies