Natural killer cells efficiently target multiple myeloma clonogenic tumor cells

The multiple myeloma (MM) landscape has changed in the last few years, but most patients eventually relapse because current treatment modalities do not target clonogenic stem cells, which are drug-resistant and can self-renew. We hypothesized that side population (SP) cells represent myeloma clonogenic stem cells and, searching for new treatment strategies, analyzed the anti-myeloma activity of natural killer (NK) cells against clonogenic cells. Activated and expanded NK cells (NKAE) products were obtained by co-culturing NK cells from MM patients with K562-mb15-41BBL cell line and characterized by flow cytometry. Functional experiments against MM cells were performed by Eu-TDA release assays and methylcellulose clonogenic assays. Side population was detected by Dye Cycle Violet labeling and then characterized by flow cytometry and RNA-Seq. Self-renewal capacity was tested by clonogenic assays. Sorting of both kind of cells was performed for time-lapse microscopy experiments. SP cells exhibited self-renewal potential and overexpressed genes involved in stem cell metabolism. NK cells from MM patients exhibited dysregulation and had lower anti-tumor potential against clonogenic cells than healthy donors’ NK cells. Patients’ NK cells were activated and expanded. These cells recovered cytotoxic activity and could specifically destroy clonogenic myeloma cells. They also had a highly cytotoxic phenotype expressing NKG2D receptor. Blocking NKG2D receptor decreased NK cell activity against clonogenic myeloma cells, and activated NK cells were able to destroy SP cells, which expressed NKG2D ligands. SP cells could represent the stem cell compartment in MM. This is the first report describing NK cell activity against myeloma clonogenic cells.

Mono-institutional phase 2 study of innovative Stereotactic Body RadioTherapy targeting PArtial Tumor HYpoxic (SBRT-PATHY) clonogenic cells in unresectable bulky non-small cell lung cancer: profound non-targeted effects by sparing peri-tumoral immune microenvironment

Background Radiotherapy-induced lymphopenia may be limiting the success of therapy and could also negatively affect the ability of immune system in mediating the bystander (BE) and abscopal effects (AE). A novel SBRT-based PArtial Tumor irradiation of HYpoxic clonogenic cells (SBRT-PATHY) for induction of the tumoricidal BE and AE by sparing the peritumoral immune microenvironment and regional circulating lymphocytes has been developed to enhance the radiotherapy therapeutic ratio of advanced lung cancer. The aim of this retrospective review of prospectively collected mono-institutional phase 2 study was to compare the outcomes between unconventional SBRT-PATHY and standard of care in unresectable stage IIIB/IV bulky NSCLC. Materials and methods Sixty patients considered inoperable or unsuitable for radical radio-chemotherapy were enrolled and treated using the following 3 regimens: SBRT-PATHY (group I, n = 20 patients), recommended standard of care chemotherapy (group II, n = 20 patients), and institutional conventional palliative radiotherapy (group III, n = 20 patients). Results Median follow-up was 13 months. The 1-year overall survival was 75, 60, and 20% in groups 1, 2 and 3, respectively (p = 0.099). The 1-year cancer specific survival was 90, 60, and 20% in groups 1, 2, and 3, respectively (p = 0.049). Bulky tumor control rate was 95% for SBRT-PATHY compared with 20% in the other two groups. BE and AE were seen by SBRT-PATHY in 95 and 45% of patients, respectively. Multi-variate analysis for cancer specific survival was significant for treatment effect with SBRT-PATHY (p < 0.001) independent of age, sex, performance status, histology, stage, treated bulky site and tumor diameter. SBRT-PATHY resulted in lower toxicity (p = 0.026), and improved symptom control (p = 0.018) when compared to other two treatment options. Conclusion SBRT-PATHY improved treatment outcomes in unresectable NSCLC and should be investigated in larger trials. Present study has been retrospectively registered on 8th of August 2019 by the ethic committee for Austrian region „Kärnten “in Klagenfurt (AUT), under study number A 31/19.

STAT1 Transcriptional Response Predicts Molecular Responses of PB-Derived Clonogenic Cells from MPN Patients to Interferon Alpha

Introduction: Interferon alpha (IFNa) is a cytokine with anti-viral and anti-tumoral properties which, either in its unmodified or pegylated form, is successfully used in the treatment of patients with myeloproliferative neoplasms (MPN), including polycythemia (PV), essential thrombocythemia (ET) and early primary myelofibrosis (PMF). Despite its efficacy including molecular responses and thus its potential disease modifying capabilities, IFNa carries the risk of significant adverse events, including liver toxicity, autoimmunity, and depression. This has prompted the development of interferons with improved features including better tolerability. However, biomarkers for response have been lacking, mostly due to the heterogeneity of cells analyzed and the difficulty in obtaining them from the bone marrow. Methods: Therefore, we have set up a clonogenic assay from Ficoll-isolated mononuclear cells of the peripheral blood of patients (PBMC) with MPN (n=51, including 17 PV, 14 ET, and 14 PMF, 1 MDS/MPN, 2 Post-PV-MF, 2 Post-ET-MF, 1 MPNu) to analyze the in vitro IFNa response (either using 0.5 µg/ml ropeginterferon alfa-2b [ropegIFNa] or 500 U/ml recombinant human IFN-alpha2b [hIFNa] or no treatment control) in the cells that drive malignant clonogenic growth in the patients. After 10-14 days, the number of colonies was assessed. For each patient and condition, twenty-five of the resulting colonies were then genotyped for zygosity of JAK2V617F and three colonies per condition were analyzed for STAT1 RNA expression using RT-qPCR, an important transcriptionally induced IFNa target gene. The number of mutated alleles was determined (zero for wildtype, one for heterozygous, two for homozygous colonies), and, based upon the change in mutant alleles after in vitro hIFNa treatment, samples were categorized into responders (mutant alleles decreased) and non-responders (mutant alleles unchanged or increased). All patients provided written informed consent, and the study was approved by the local ethics committee. Results: To assess potential differences between the two interferons used, a 14-day proliferation assay was performed in Set-2 cells, showing more sustained inhibition of proliferation by ropegIFNa than hIFNa (p=0.007), confirming the longer half-life of ropegIFNa. In the clonogenic assay, different colonies were observed depending on the MPN subtype, ranging from exclusive CFU-E (PV) to exclusive CFU-G and CFU-M (PMF) types of colonies. Both hIFNa and ropegIFNa significantly inhibited colony growth as compared to the control and reduced the colony number to 72.7 % (hIFNa) and 58.5% (ropegIFNa) of the untreated control, with ropegIFNa showing significantly stronger effects than hIFNa (p=0.0137). Interestingly, there were marked differences in the amount of JAK2 alleles in the colonies between the patients (n=24 analyzed), with 16 patients showing a reduction of up to 22% of the allele burden upon in vitro treatment with hIFNa (responders), while 8 patients showed no reduction or even an increase of up to 15% (non-responders) (p=0.0001). Basal STAT1 expression in the colonies (three colonies per patient and treatment) was significantly lower in responders than non-responders (p=0.0200). After treatment with hIFNa, STAT1 expression was induced to similar levels in both responders and non-responders (p=0.6620). As a result, STAT1 fold induction was significantly higher in responders than non-responders (p=0.0013). Interestingly, there was no correlation of the responses with current clinical treatment of the patients (previous interferon exposure did not prevent responses) or with the MPN subtype, confirming clinical reports that patients with myelofibrosis can respond to interferon. Conclusion: In conclusion, clonogenic cells from the peripheral blood of MPN patients can be used to assess their molecular response to IFNa. In vitro, ropegIFNa induced stronger effects than non-pegylated IFNa. The responses were heterogeneous at the molecular level, but, when combined with RNA expression analysis, we were able to dissect molecular responders from non-responders. The mechanisms for these differences and their clinical impact are currently being studied. Disclosures Gezer: AMGEM: Membership on an entity's Board of Directors or advisory committees. Isfort:Mundipharma: Other: Travel reimbursement; Amgen: Other: Travel reimbursement; Hexal: Other: Travel reimbursement; BMS: Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement; Roche: Other: Travel reimbursement; Alexion: Other: Travel reimbursement. Brümmendorf:Ariad: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment; Novartis: Consultancy, Research Funding; Merck: Consultancy. Koschmieder:Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Foundation: Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

The Capacity of Long-Term in Vitro Proliferation of Acute Myeloid Leukemia Cells Supported Only by Exogenous Cytokines Is Associated with a Patient Subset with Adverse Outcome

Acute myeloid leukemia (AML) is an aggressive malignancy, which is highly heterogeneous with regard to chemosensitivity and biological features. The AML cell population is organized in a hierarchy that is reflected in the in vitro growth characteristics, with only a minority of cells being able to proliferate for more than two weeks. In this study, we investigated the ability of AML stem cells to survive and proliferate in suspension cultures in the presence of exogenous mediators but without supporting non-leukemic cells. We saw that a high number of maintained stem cells (i.e., a large number of clonogenic cells after five weeks of culture) was associated with decreased overall survival for patients receiving intensive chemotherapy; this prognostic impact was also detected in the multivariate/adjusted analysis. Furthermore, the patients with many clonogenic cells presented more frequently with mutations in transcription-related genes, and also showed a higher abundance of proteins involved in transcription at the time of diagnosis. In conclusion, the growth characteristics of the long-term proliferating leukemic stem cells seem to have an independent prognostic impact in human AML, and these characteristics appear to be reflected by the mutational landscape and the proteome of the patients at the time of diagnosis.

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomasin vivoand thereby affect treatment plans, and they call for further investigations.

Oncogene-dependent survival of highly transformed cancer cells under conditions of extreme centrifugal force – implications for studies on extracellular vesicles

Extracellular vesicles (EVs), including exosomes, are a subject of intense interest due to their emission by cancer cells and role in intercellular communication. Earlier reports suggested that oncogenes, such as RAS, MET or EGFR, drive cellular vesiculation. Interestingly, these oncogenes may also traffic between cells using the EV-mediated emission and uptake processes. One of the main tools in the analysis of EVs are ultracentrifugation protocols designed to efficiently separate parental cells from vesicles through a sequence of steps involving increasing g-force. Here we report that ultracentrifugationonly EV preparations from highly transformed cancer cells, driven by the overexpression of oncogenic H-ras (RAS-3) and v-src (SRC-3), may contain clonogenic cancer cells, while preparations of normal or less aggressive human cell lines are generally free from such contamination. Introduction of a filtration step eliminates clonogenic cells from the ultracentrifugate. The survival of RAS-3 and SRC-3 cells under extreme conditions of centrifugal force (110,000 g) is oncogene-induced, as EV preparations of their parental non-tumourigenic cell line (IEC-18) contain negligible numbers of clonogenic cells. Moreover, treatment of SRC-3 cells with the SRC inhibitor (PP2) markedly reduces the presence of such cells in the unfiltered ultracentrifugate. These observations enforce the notion that EV preparations require careful filtration steps, especially in the case of material produced by highly transformed cancer cell types. We also suggest that oncogenic transformation may render cells unexpectedly resistant to extreme physical forces, which may affect their biological properties in vivo.

Effects Of Long-Term Exposure To Phytochemicals and Nsaids On The Self-Renewal Of Leukemic Blast Progenitors In Vitro

Objective The preventive and therapeutic roles of non-steroid anti-inflammatory drugs (NSAIDs) and resveratrol (RSV) is well established. The preventive role of NSAIDs and aspirin, in particular, in colorectal cancer is well established. More recently, it has been suggested that aspirin may also have a therapeutic role. RSV, a polyphenol found in grape skins, has been proposed to reduce the risk of cancer development. Additionally, these studies indicate that resveratrol's chemoprevention effect is dose and duration dependent. The anti-cancer effects of ascorbic acid (vitamin C, Vit-C) have also been reported. However, the molecular mechanisms involved in NSAIDs- and these phytochemical-mediated tumor suppressing activities are not yet completely defined. We investigated the continuous additive effects of RSV, Vit-C, indomethacin (IND), and NS-398 on the growth of leukemic blast clonogenic cells in methylcellulose and the cumulative clonogenic cells in liquid suspension for up to one month by serial replating of leukemic cell lines. The interactive effects of additives were also investigated. Methods Myeloid (HL-60, K-562, MO7-E and U-937) and B lymphoid (Daudi, Raji and U-266) cell lines were used. RSV, Vit-C, IND, and NS-398 were added to the culture at a final concentration of 10, 300, 30, and 30μM, respectively. The concentrations of the additives in any of the cell lines were lower than IC50. Proportion of senescence was determined by using Senescence Detection Kit. Results Vit-C, RSV, IND and NS-398 significantly inhibited colony formation in three (43%), three (43%), three (43%) and four (57%) cell lines, respectively. In contrast, NS-398 significantly stimulated colony formation in U-937. Interactive effects between RSV/ Vit-C were synergistic (S) in four (57%), offset (O) in two (29%), and additive (A) in one (14%), those between RSV/IND were S in four (57%) and O in three (43%), and those between RSV/NS-398 were S in four (57%), O in two (29%) and A in one (14%), respectively. Logarithmic linear increase in the number of cumulative clonogenic cells in suspension was noted in any of the cell lines. RSV completely abrogated and partially but significantly inhibited the growth in three myeloid cell lines (MO7-E, U-937 and K-562) and four remaining lymphoid and myeloid cell lines, respectively. When RES was removed from the culture, complete recovery of the growth was noted. Vit-C, IND, and NS-398 only partially but significantly inhibited the growth in two (29%), one (14%) and one (14%) cell lines, respectively. In any of the cell lines, there was no correlation between the inhibitory effects of the additives on the clonogenic cell growth in methylcellulose and those in liquid suspension. RES enhanced p14 and/or p21 expression. Senescence was induced in all cell lines, except for Raji. RES induced significantly high proportion of senescence in MO7-E and K-562 after two-week culture comapred with control. Conclusions Phytochemicals and NSAIDs showed differential effects on colony formation in methylcellulose and cumulative clonogenic cell growth in suspension, probably owing to the different actions of these compounds in differentiation and self-renewal of these cell lines. Disclosures: No relevant conflicts of interest to declare.