RESUMEN
Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24(+)/CD44(+), CD133(+), and aldehyde dehydrogenase(bright)) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.
Asunto(s)
Antígenos CD/inmunología , Inmunidad Celular , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Células Madre Neoplásicas/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos NOD , Neoplasias/patología , Células Madre Neoplásicas/patologíaRESUMEN
BACKGROUND: Increasing studies implicate cancer stem cells (CSCs) as the source of resistance and relapse following conventional cytotoxic therapies. Few studies have examined the response of CSCs to targeted therapies, such as tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have differential effects on CSC populations depending on their mechanism of action (anti-proliferative vs. anti-angiogenic). METHODS: We exposed human sarcoma cell lines to sorafenib, regorafenib, and pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray (TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and non-parametric statistical analyses were performed as appropriate. RESULTS: After functionally validating the CSC phenotype of ALDHbright sarcoma cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell lines (P < 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright cells from baseline (P < 0.05). In contrast, we observed negligible effects on viability and CSC sub-populations with pazopanib. At low doses, there was progressive CSC enrichment in vitro after longer term exposure to sorafenib although the anti-proliferative effects were attenuated. In vivo, sorafenib improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells 2.5 - 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib and regorafenib with a corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects from pazopanib were observed. TMA analysis of archived specimens from sarcoma patients treated with sorafenib demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas clinical specimens obtained longitudinally from a patient treated with pazopanib showed no enrichment for ALDHbright cells (P > 0.05). CONCLUSIONS: Anti-proliferative TKIs appear to enrich for sarcoma CSCs while anti-angiogenic TKIs do not. The rational selection of targeted therapies for sarcoma patients may benefit from an awareness of the differential impact of TKIs on CSC populations.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Sarcoma/tratamiento farmacológico , Familia de Aldehído Deshidrogenasa 1 , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antígenos CD/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Indazoles , Isoenzimas/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Ratones Endogámicos NOD , Terapia Neoadyuvante , Células Madre Neoplásicas/efectos de los fármacos , Niacinamida/farmacología , Niacinamida/uso terapéutico , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Retinal-Deshidrogenasa/metabolismo , Sarcoma/secundario , Sorafenib , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Short interfering RNAs (siRNAs) are promising drug candidates for a wide range of targets including those previously considered "undruggable". However, properties associated with the native RNA structure limit drug development, and chemical modifications are necessary. Here we describe the structure-guided discovery of functional modifications for the guide strand 5'-end using computational screening with the high-resolution structure of human Ago2, the key nuclease on the RNA interference pathway. Our results indicate the guide strand 5'-end nucleotide need not engage in Watson-Crick (W/C) H-bonding but must fit the general shape of the 5'-end binding site in MID/PIWI domains of hAgo2 for efficient knockdown. 1,2,3-Triazol-4-yl bases formed from the CuAAC reaction of azides and 1-ethynylribose, which is readily incorporated into RNA via the phosphoramidite, perform well at the guide strand 5'-end. In contrast, purine derivatives with modified Hoogsteen faces or N2 substituents are poor choices for 5'-end modifications. Finally, we identified a 1,2,3-triazol-4-yl base incapable of W/C H-bonding that performs well at guide strand position 12, where base pairing to target was expected to be important. This work expands the repertoire of functional nucleotide analogues for siRNAs.
Asunto(s)
Proteínas Argonautas/metabolismo , Conformación de Ácido Nucleico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Proteínas Argonautas/química , Secuencia de Bases , Sitios de Unión , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Nucleótidos/química , Compuestos Organofosforados/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Triazoles/químicaRESUMEN
Currently, there is no consensus on best practices to prevent hemorrhagic cystitis (HC) in patients receiving posttransplant cyclophosphamide (PTCy). We retrospectively reviewed 194 patients undergoing their first hematopoietic cell transplant (HCT) who received PTCy from 2014 to 2018 to describe the incidence and severity of HC, identify potential risk factors, and impact of HC on HCT outcomes. Standard HC prophylaxis was hyperhydration with forced diuresis and mesna at 320% the daily dose of PTCy. Incidence of HC was 31.4% at day +100 of HCT. Median onset of HC was 12 days with 11.5% grade 3 HC and no Grade 4 HC. Patients with chemical HC experienced earlier onset (7 days vs. 34 days, p < 0.001) with a shorter median resolution time (5 days vs. 14 days, p = 0.001) when compared to BK-associated HC. In multivariate analysis, age above 60 years (HR 4.16, p = 0.006) and myeloablative conditioning (HR 2.44, p = 0.054) were associated with higher risk for HC, but overall, HC did not affect nonrelapse mortality or overall survival. In conclusion, hyperhydration with forced diuresis combined with aggressive mesna dosing is an effective strategy in preventing severe PTCy-associated HC, subsequently preventing any negative impact on transplant outcome.
Asunto(s)
Cistitis , Trasplante de Células Madre Hematopoyéticas , Ciclofosfamida/efectos adversos , Cistitis/epidemiología , Cistitis/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Incidencia , Mesna/efectos adversos , Persona de Mediana Edad , Estudios Retrospectivos , Trasplante HomólogoRESUMEN
Natural killer (NK) cells are innate lymphocytes postulated to mediate resistance against primary haematopoietic but not solid tumor malignancies. Cancer stem cells (CSCs) are a small subset of malignant cells with stem-like properties which are resistant to chemo- and radiotherapies and are able to repopulate a tumor after cytoreductive treatments. We observed increased frequencies of stem-like tumor cells after irradiation, with increased expression of stress ligands on surviving stem-like cells. Ex vivo NK cells activated by low dose IL2 in vitro and IL15 in vivo displayed an increased ability to target solid tumor stem-like cells both in vitro and in vivo after irradiation. Mechanistically, both upregulation of stress-related ligands on the stem-like cells as well as debulking of non-stem populations contributed to these effects as determined by data from cell lines, primary tumor samples, and most relevant patient derived specimens. In addition, pretreatment of tumor-bearing mice with local radiation prior to NK transfer resulted in significantly longer survival indicating that radiation therapy in conjunction with NK cell adoptive immunotherapy targeting stem-like cancer cells may offer a promising novel radio-immunotherapy approach in the clinic.