Association of extensive RNA disruption with natural killer cell-mediated death of K562 chronic myelogenous leukemia cells

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Pritzker, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2944450/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Extensive degradation of tumour 28S and 18S ribosomal RNAs, coupled with the accumulation of ribosomal RNA degradation products, is associated with pathologic complete response and improved disease-free-survival in breast cancer patients. Various chemotherapy agents and cellular stressors are known to trigger this process, termed ‘RNA disruption’, in tumour cells. However, it’s unclear whether immunotherapies, with or without chemotherapy administration, also trigger RNA disruption. To address this question, we assessed the ability of natural killer (NK) cells to induce RNA disruption and cell death in K562 chronic myeloid leukemia cells in vitro . We found that NK cells strongly stimulated RNA disruption, cytotoxicity (loss of plasma membrane integrity) and cell death (generation of cells with a subG1 DNA content) in K562 cells. Pre-activation of NK cells with interleukin-2 or pre-treatment of K562 cells with the chemotherapy drug doxorubicin augmented RNA disruption in K562 cells. RNA degradation patterns looked very similar between NK cell-treated and doxorubicin-treated K562 cells. Our observations suggest that RNA disruption is strongly associated with cell death irrespective of the death-inducing stimulus and raise the prospect that tumour RNA disruption may be a useful biomarker for quantifying cancer patients’ response to immunotherapies, with or without co-administration of chemotherapy drugs. Biological sciences/Biological techniques Biological sciences/Cancer Biological sciences/Immunology Biological sciences/Molecular biology Health sciences/Biomarkers Health sciences/Molecular medicine Health sciences/Oncology RNA disruption ribosomal RNA degradation chemotherapy immunotherapy natural killer cells plasma membrane integrity cell death DNA content Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The majority of breast cancer patients undergoing chemotherapy and/or immunotherapy treatments often suffer from toxic side effects from these treatments, including cardiotoxicity, thromboemboli, pneumonitis, hepatitis, neurotoxicities, neutropenia, and secondary cancers 1 – 6 . In contrast, only a minority of such patients receive a substantial survival benefit from treatment, in particular for some tumour subtypes 1,2,7−9 . Consequently, there is growing interest in treatment response assessment tools that can predict the outcome of regimens involving standard cytotoxic drugs with or without the employment of immunotherapies 10 . With such tools, oncologists could determine whether their patients are responding positively during the early stages of treatment. Patients with non-responsive tumours could then forgo the costs and toxicities associated with ineffective regimens and proceed to alternate treatments. A treatment response assessment tool showing promise is the RNA disruption assay (RDA). The RDA quantifies a specific phenomenon we termed ‘RNA disruption’, which is characterized by the degradation of the 28S and 18S ribosomal RNAs (rRNAs) with the concomitant accumulation of abnormal bands between the 28S and 18S rRNAs on RNA electropherograms. These bands are derived, at least in part, from the 28S rRNA 11 . In a sub-study of the NCIC-CTG MA.22 12 and NeoAva 13 clinical trials, we demonstrated that high mid-treatment tumour RNA disruption was associated with little to no residual disease post-treatment (as indicated by a pathologic complete response [pCR] or no residual cancer burden) and increased survival of breast cancer patients following neoadjuvant chemotherapy, suggesting that the RDA can predict treatment outcome much earlier than standard post-treatment evaluations. The assay was also able to predict response and disease-free survival after CHOP chemotherapy in canine lymphoma patients 14 . The predictive value of the RDA in neoadjuvant chemotherapy is currently being investigated in a large international clinical trial called ‘BREVITY’, involving approximately 600 female patients with advanced breast cancer ( https://clinicaltrials.gov/ct2/show/NCT03524430 ). RNA disruption has been observed in vitro in a wide variety of tumour and non-tumour cell types, including breast epithelial cells, ovarian endometrioid cells, myeloid cells, melanocytes and vascular endothelial cells 11 . It is triggered by many structurally and mechanistically distinct chemotherapy agents as well as specific cell stressors, including endoplasmic reticulum stress, oxidative stress and nutrient/growth factor limitation 11 . This phenomenon is associated with cell death in vitro , as measured by a reduction in cell numbers, loss of cell replicative capacity, and the generation of cells with a subG1 DNA content 11 . Although RNA disruption was found to be associated with chemotherapy-induced tumour cell death in vitro 11 , 15 , 16 and clinical response to neoadjuvant chemotherapy in cancer patients in vivo 14 , 17 , 18 , its occurrence in response to an immunogenic challenge has yet to be explored. Immune checkpoint inhibitors represent one of the most promising new classes of chemotherapy drugs in oncology 19 . These drugs strongly augment the ability of a patient’s own immune cells to recognize and destroy tumours by inhibiting their specific immunoevasion pathways used to block immune cell killing mechanisms 20 . An example of an immunoevasion mechanism is the binding of programmed death ligand 1 (PDL1) on the surface of tumour cells to the programmed cell death 1 (PD1) receptor on the surface of helper T cells, which inhibits the activation of the latter cells. This blocks the production of cytokines by T cells, which would otherwise activate cytotoxic T cells. Blocking T cell activation also prevents their release of interleukin (IL)-2 and other cytokines that promote B cell proliferation 21 , B cell differentiation into antibody-secreting plasma cells 22 , and activation of natural killer (NK) cells 23 . Thus, PDL1 inhibitory drugs, such as pembrolizumab, can restore the ability of the immune system to recognize and kill tumours 20 . Interestingly, PDL1 inhibitors have resulted in dramatic improvements in the treatment of certain cancers including lung cancer 24 , 25 , renal cell carcinoma 26 and melanoma 27 . Moreover, pembrolizumab was found to strongly augment the pCR rate for breast cancer patients treated with neoadjuvant docetaxel chemotherapy in the I-SPY2 clinical trial 28 . Other immunomodulatory approaches, such as chimeric antigen receptor (CAR) T cell therapy, can effectively treat a variety of haematological malignancies and solid tumours. In these approaches, T cells are taken from the patient and genetically engineered to give them the ability to target specific receptors on the surface of tumour cells, such as CD19 in B cell-derived cancers and CD30 in Hodgkin lymphoma 29 – 31 . Unfortunately, the above immunotherapies have significant toxicities, including cytokine-release syndrome 32 , 33 , neurotoxicities 32 , 34 and cardiotoxicities 33 (associated with CAR-T therapy), as well as pneumonitis, neuropathies, endocrinopathies, hepatitis and dermatitis (associated with PD1/PDL1 inhibitors) 35 . As is the case with chemotherapy, only a limited number of patients respond favorably to immunotherapy. For example, < 20% of lung cancer patients and < 45% of patients with malignant melanoma respond to PDL1 inhibitors 36 , 37 . Similarly, recent reviews indicate that objective and complete response rates to CAR T cell therapy vary widely from study to study 38 – 40 . Given the large number of non-responding patients, and the costs and toxicities associated with the above immunotherapies, a biomarker that could effectively predict treatment outcome prior to treatment or early during treatment would be highly valued. In this study, we began exploring the potential utility of the RDA to quantify the response of cancer patients to immunotherapy by first determining whether immune cells can trigger RNA disruption in tumour cells in vitro , and whether this disruption is accompanied by cytotoxicity and a loss of tumour cell viability. We report here that freshly isolated NK cells from healthy volunteers strongly induce RNA disruption, cytotoxicity (as measured by a reduction in plasma membrane integrity) and cell death (as measured by the generation of cells with a subG1 DNA content) in human chronic myeloid leukemia K562 cells. Moreover, pre-activation of NK cells with IL-2 or pre-treatment of K562 cells with doxorubicin augments NK cell-dependent RNA disruption in K562 cells. Taken together with our previously published findings, our work suggests that RNA disruption is strongly associated with tumour cell death, regardless of the death-inducing agent. Results Freshly isolated human NK cells induce RNA disruption in human chronic myeloid leukemia cells. To assess whether immune cells can induce RNA disruption in tumour cells, we first isolated peripheral blood mononuclear cells (PBMCs) from healthy human volunteers, and enriched the cell preparation for NK cells. We then treated K562 human chronic myeloid leukemia cells with freshly isolated NK cells for 4 h, and measured the extent of RNA disruption occurring in K562 cells using the RDA. To ensure that RNA from NK cells did not contribute to the signal, we removed NK cells from co-cultures prior to RNA extractions using selection on CD56 + magnetic beads (see Methods). We found that NK cells triggered RNA disruption in K562 cells, particularly when they outnumbered their target cells (NK:K562 ratios of 2:1 and 4:1) (Figs. 1 a and 1 b, compare lanes/columns 10 and 12 to lane/column 4). We noted pronounced reductions in the intensities of the 28S and 18S rRNA bands, and increased intensities of abnormal bands in the “inter-region” between the 28S and 18S rRNA bands of electropherograms (Fig. 1 a). These changes in RNA banding patterns were reflected in increased RNA disruption indexes (RDIs) (Fig. 1 b), which express the extent of RNA disruption as a ratio between the combined areas of the abnormal peaks and the combined areas of the intact 28S and 18S rRNA bands on RNA electropherograms 17 . Interestingly, we also found that the extent of RNA disruption progressively increased as NK cells became more abundant than their target cells (Figs. 1 a and 1 b). We observed little to no RNA disruption in K562 cells when NK cells equaled or were outnumbered by K562 cells (NK:K562 ratios of 1:1 and 0.5:1, respectively) (Figs. 1 a and 1 b, compare lanes/columns 6 and 8 to lane/column 4). Few bands were noted in the inter-region of electropherograms in these instances (Fig. 1 a), and relatively low RDI values were consistently calculated (Fig. 1 b). We also noted an interesting negative association between RNA disruption and RNA abundance within K562 cells. In general, as RNA disruption became more prevalent in K562 cells, RNA abundance within cells (as measured by spectrophotometry) progressively decreased (Fig. 1 ), suggesting that NK cells also cause a reduction in RNA levels with their target cells, likely due to cell death/destruction (Fig. 4 ). To confirm the absence of NK cell RNA from K562 RNA preparations, we cultured NK cells in the absence of K562 cells, removed the NK cells from cultures using CD56 + selection, and isolated and analysed total RNA from what remained. We recovered low to negligible amounts of RNA from these “NK cells only” controls (< 10 ng µL − 1 ) (Fig. 1 c, columns 5, 7, 9 and 11), and noted only weak, barely detectable bands on RNA electropherograms (Fig. 1 a, lanes 5, 7, 9 and 11). Furthermore, the recovered RNA was deemed ‘not assessable’ by the RDA owing to insufficient RNA concentration to detect the 28S and 18S rRNA peaks (Fig. 1 b, columns 5, 7, 9 and 11). Together, these findings suggest that NK cells were effectively removed from the NK/K562 co-cultures, and that NK cell-derived total RNA did not contribute substantially to the K562 cell RNA electropherogram banding pattern or to RDI measurements. Because CD56 + selection was required to remove NK cells from co-cultures prior to RDA analyses, we determined the impact of the selection process itself on RNA disruption in K562 cells cultured in the absence of NK cells. Although the selection process did not impact RDI values (Fig. 1 b, compare column 4 to column 3), we did note that untreated K562 cells subjected to CD56 + selection exhibited lower-molecular-weight abnormal bands in the inter-region of RNA electropherograms than untreated K562 controls that did not undergo this selection (Fig. 1 a, compare lane 4 to lane 3). We also found that the concentration of RNA isolated from selected K562 cells was lower than that of unselected cells (Fig. 1 c, compare column 4 to column 3), suggesting that the NK cell removal procedure likely resulted in the loss of some K562 cells, which is not unexpected with lengthy procedures. This indicates that the NK cell removal procedure had a slight effect on K562 RNA levels and banding pattern. We included 0-h controls in all of our experiments to provide a reference point as to the state of RNA disruption in K562 cells prior to treatment initiation. Untreated K562 cells (at 0 h) generally demonstrated slightly greater levels of abnormal bands in the inter-region of the RNA electropherogram and higher RDI values than untreated cells after 4 h of incubation (Figs. 1 a and 1 b, compare lane/column 2 to lane/column 4). This was likely due to stress experienced by K562 cells during processing prior to incubation, which was relieved upon incubation in high-nutrient cell culture medium containing growth factors. Pre-treatment of NK cells with IL-2 augments NK cell-mediated RNA disruption in K562 cells. IL-2 is a cytokine known to increase NK cell-mediated tumour cytotoxicity 41 , 42 . To determine whether pre-treatment of NK cells with IL-2 could enhance their ability to induce RNA disruption in K562 cells, we repeated the above experiments using NK cells pre-treated with different concentrations of IL-2. We observed more extensive disruption in K562 cells treated with IL-2-stimulated NK cells than in K562 cells treated with unstimulated NK cells, particularly when NK cells were equally (NK:K562 ratio of 1:1) or more abundant (NK:K562 ratios of 2:1) than their target cells (Figs. 2 a and 2 b, compare lanes/columns 9 and 10 to lane/column 8; lanes/columns 12 and 13 to lane/column 11; and lanes/columns 15 and 16 to lane/column 14). Furthermore, we again found a negative association between RNA disruption and RNA yields, with K562 cells treated with pre-activated NK cells generally yielding less concentrated RNA extracts than K562 cells treated with unstimulated NK cells (Fig. 2 c, compare columns 9 and 10 to column 8; columns 12 and 13 to column 11; and columns 15 and 16 to column 14). Taken together, our findings suggest that pre-activation of NK cells with IL-2 enhances NK cell-mediated RNA disruption and reduces total RNA content in K562 cells in a dose-dependent manner. We confirmed the successful removal of pre-activated NK cells from co-cultures prior to RNA isolation from K562 cells as above. Here, we tested only the highest density NK cell mono-culture (corresponding to cell amounts found in co-cultures with an NK:K562 ratio of 2:1), pre-treated or not with IL-2. We reasoned that if the largest number of NK cells used to treat K562 cells was effectively removed during CD56 + positive selection, then the smaller amounts of NK cells in the co-cultures with lower NK:K562 ratios would also have been successfully removed. We recovered little to no RNA from the “NK cells only” controls (< 10 ng µL − 1 ), irrespective of the IL-2 dose used to pre-activate NK cells (Fig. 2 c, columns 5, 6 and 7), and what was recovered was deemed un-assessable by the RDA owing to insufficient RNA (Fig. 2 b, columns 5, 6 and 7). Overall, these controls revealed that NK cells were effectively removed from co-cultures, and there was little contribution of NK cell RNA towards electropherograms of RNA isolated from purified K562 cells. NK cells pre-incubated with or without IL-2 induce loss of membrane integrity in K562 tumour cells. An earlier study found that freshly isolated NK cells are cytotoxic to K562 cells 43 . To explore this further, we co-cultured K562 cells with unstimulated NK cells for 4 h, and monitored the loss of plasma membrane integrity using a live-cell imaging and analysis system with a live/dead cell-labelling assay. As the plasma membrane is responsible for protecting the cell from its surroundings and for transporting essential substances in and out of the cell, loss of its integrity often precedes cell death 44 . Thus, loss of plasma membrane integrity is commonly used as a marker of immunocytotoxicity 45 , 46 . We found that the number of K562 cells with a compromised plasma membrane (normalized to total live cells) increased when K562 cells were co-cultured with NK cells (Fig. 3 a, compare columns 3–6 to column 2), suggesting that NK cells trigger a loss of membrane integrity in K562 cells. Furthermore, the number of K562 cells exhibiting loss of membrane integrity increased with increasing numbers of co-incubated NK cells (Fig. 3 a). To explore the effect of IL-2 pre-activation on NK cell-mediated loss of K562 cell membrane integrity, we repeated our experiments using NK cells pre-treated overnight with IL-2. We detected a greater number of membrane-compromised K562 cells in co-cultures containing IL-2-stimulated NK cells than in co-cultures containing unstimulated NK cells, with numbers increasing with increasing IL-2 doses (Fig. 3 b, compare columns 4 and 5 to column 3; columns 7 and 8 to column 6; and columns 10 and 11 to column 9), suggesting that IL-2 activation promotes NK cell-dependent cytotoxicity towards K562 cells. While conducting these experiments, the imaging system’s parameters were adjusted to exclude counting of labelled NK cells. To ensure that labelled NK cells in co-cultures were not counted in error by the imaging system, we performed the live/dead cell-labelling assay using NK cells (with or without prior IL-2 activation) cultivated in the absence of K562 cells. We found that the amount of labelled NK cells counted was negligible (Supplementary Figs. S2-S5). These findings confirm that NK cells were effectively filtered out by the imaging system, and that counts of membrane-compromised cells in co-cultures stemmed exclusively from K562 cells. NK cells promote the generation of non-viable K562 tumour cells with a subG1 DNA content. Cells with a subG1 DNA content are non-viable as they lack sufficient genetic material to maintain cellular processes 47 , thus making the increased detection of cells with a subG1 DNA content a robust marker of cell death. Given NK cells’ impact on the plasma membrane of K562 cells (Fig. 3 ), we next assessed the ability of NK cells to increase the abundance of K562 cells with a subG1 DNA content. We co-cultured NK cells with K562 cells at different NK:K562 ratios for 4, 24 and 48 h. After removing the NK cells from co-cultures, K562 cells were fixed and stained with propidium iodine. The percentage of K562 cells with a subG1 DNA content was then determined using flow cytometry. We found a greater percentage of K562 cells had a subG1 DNA content when co-cultured with NK cells than when grown in the absence of NK cells (Fig. 4 , compare columns 3–6 to column 2; Supplementary Fig. S6). The extent of the increase in the population of K562 cells with a subG1 DNA content was more pronounced as the abundance of NK cells increased relative to their target cells (Fig. 4 , compare columns 3–6 to column 2; Supplementary Fig. S6). The length of the incubation period increased the population of K562 cells with a subG1 DNA content when the NK:K562 ratio was very high (4:1) (Fig. 4 , compare for example column 6 between panels; Supplementary Fig. S6). Taken together, our findings suggest that NK cells promote the generation of non-viable K562 cells with a subG1 DNA content in a cell number- and time-dependent manner. RNA disruption coincides with immunocytotoxicity and cell death in NK cell-treated K562 cells. Our earlier work demonstrated that RNA disruption coincides with cytotoxicity and cell death in tumour cells treated with chemotherapeutic drugs 11 . We thus hypothesized that this relationship holds true in NK cell-treated K562 cells. We compared NK cell-mediated RNA disruption in K562 cells (quantified using the RDA) (Fig. 1 ) with K562 cell immunocytotoxicity (measured as a loss of plasma membrane integrity using live/dead cell labelling) (Fig. 3 ) and K562 cell death (measured as the generation of K562 cells with a subG1 DNA content using flow cytometry) (Fig. 4 , Supplementary Fig. S6). Pre-treatment of K562 cells with doxorubicin enhances NK cell-mediated RNA disruption in K562 cells. An earlier study looking at the effect of chemotherapeutic drugs on tumour cells found that doxorubicin triggers RNA disruption in K562 cells 11 . Given that immunotherapy drugs like pembrolizumab have been shown to improve outcome from chemotherapy in early and late stage triple negative breast cancer 48 , 49 and in metastatic non-small cell lung cancer 50 , and they are commonly prescribed along with chemotherapy, we next investigated whether we could improve NK cell-mediated RNA disruption by pre-treating K562 cells with doxorubicin prior to addition of NK cells. To this end, we pre-treated K562 cells with doxorubicin, then incubated the doxorubicin-treated K562 cells with NK cells in doxorubicin-free medium. We then removed NK cells from co-cultures using CD56 + selection, isolated total RNA from remaining intact and lysed K562 cells and quantified RNA disruption using the RDA. We found that 1 and 10 µM doxorubicin, in the absence of subsequent treatment with NK cells, triggered RNA disruption and reduced RNA abundance in K562 cells, with greater effects achieved with higher drug doses (Fig. 5 , compare lanes/columns 9 and 13 to lane/column 5). Similarly, NK cells alone promoted RNA disruption and reduced RNA levels in K562 cells that were not exposed to doxorubicin (Fig. 5 , compare lanes/columns 6–8 to lane/column 5). Interestingly, when we co-cultured doxorubicin-treated K562 cells with NK cells, we detected a dramatic increase in RNA disruption in comparison to levels seen in K562 cells treated either with doxorubicin or NK cells alone. Of particular note, we found that levels of disruption seen in K562 cells treated with both doxorubicin and NK cells far exceeded the sum of disruption seen in cells treated with only the chemotherapeutic drug or only NK cells (Figs. 5 a and 5 b, compare for example lane/column 16 to lanes/columns 8 and 13). We also detected very low levels of RNA in K562 cells treated with both doxorubicin and NK cells (Fig. 5 c, columns 10–12 and 14–16). Taken together, our findings suggest that pre-treatment of K562 cells with doxorubicin augments the ability of NK cells to induce RNA disruption and reduce RNA abundance in K562 cells. NK cells and doxorubicin generate similar RNA disurption profiles within K562 cells. Since this study was the first to investigate immune cell-mediated RNA disruption, it was of interest to compare the abnormal RNA banding patterns of K562 cells treated with doxorubicin and those of K562 cells co-cultured with NK cells. We thus compared the RNA disruption profiles of both sample types by overlaying their electropherograms. Four pairs of samples with similar RDI values were used for comparison. We observed treatment-related reductions in the 28S and 18S rRNA peaks for both NK cell-treated and doxorubicin-treated K562 cells (Fig. 6 ), compared to untreated cells (Fig. 1 a). In addition, the diffuse series of abnormal RNA peaks on electropherograms (particularly in the inter-region) appeared qualitatively similar (Fig. 6 ). Thus, regardless of contrasting mechanisms of cell death, doxorubicin and NK cells induce strikingly similar RNA disruption patterns in K562 cells. Discussion Freshly isolated human NK cells are cytotoxic to K562 cells and induce both RNA disruption and cell death in K562 cells. We have confirmed in our study that NK cells are cytotoxic to K562 cells in culture, as measured by the ability of NK cells to compromise plasma membrane integrity (Fig. 3 ). NK cells also induce the death of K562 cells, as measured by strong increases in the number of K562 cells with a non-viable subG1 content (Fig. 4 , Supplementary Fig. S6). We further show that the above phenotypic changes are accompanied by RNA disruption (higher RDI values) and reduced cellular RNA concentration (Fig. 1 ). The magnitude of all four phenotypic changes is dependent upon the number of NK cells added to K562 cell preparations (Figs. 1 , 3 and 4 ). Interestingly, the ratios of NK cells to K562 cells used to induce the above phenotypic changes were very similar to those employed in other studies. Kandarian et al. 51 developed a flow cytometric cytotoxicity assay to assess human NK cell activity towards K562 cells. Their method was very similar to our loss of membrane integrity cytotoxicity assay. K562 cells were pre-labeled with a fluorescent dye to allow for discrimination from NK cells, and injured K562 cells with a compromised plasma membrane were identified using a cell-impermeable nucleic acid stain. A 4-h incubation period was also used in their study 51 , and the NK:K562 ratios ranged from 0.625 to 5, very similar to those used in our study. Using a similar flow cytometric approach, Kwoen et al . 52 also demonstrated NK cell cytotoxicity towards K562 cells (using a 4-h incubation period and NK:K562 ratios identical to those used in this study). Other studies involving a 51 Cr release assay 53 or image cytometry 54 employed identical incubation times but higher NK:K562 ratios. The above observations strongly suggest that the RNA disruption assay required similar parameters to other assays in order to demonstrate NK cell-mediated toxicity toward K562 cells. The mechanism by which NK cells are cytotoxic to K562 cells and induce their death is not entirely clear, but appears to involve the interaction between the NK62D and Ly49D receptors on the surface of NK cells with the Rae-I and H2D receptors on K562 cells 55 . This results in the phosphorylation of motifs on the DNAX activation proteins Dap10 and Dap12 at the surface of NK cells, respectively, and subsequent activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway in NK cells. This in turn activates the transcription factors NF-κB and NFAT 55 , which ultimately promotes the release of cytokines, chemokines and granzymes from NK cells and the release of perforins from their target cells 55 . The perforins then facilitate entry of granzymes into the target cell, which activates pro-apoptotic caspases 56 . In addition, the release of cytokines promotes apoptotic cell death through an interaction of Fas ligand on the surface of NK cells with Fas receptors on the surface of target cells 55 . There is some evidence that rRNA degradation may precede or accompany cell death in cells. For example, Houge and colleagues found that 8-(4-Chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate strongly induces apoptosis in IPC-81 rat myeloid leukemia cells, and this was temporally correlated with the suppression of protein translation, internucleosomal DNA fragmentation, and the fragmentation of the 28S rRNA through cleavage at specific sites within its V3 and V13 variable regions 57 . These investigators reported similar findings in NB4 myeloid leukemia cells treated with okadaic acid, rat thymocytes treated with prednisolone, and bovine endothelial cells treated with tumour necrosis factor and cycloheximide, except that the variable regions targeted during 28S rRNA fragmentation differed depending upon the apoptosis-inducing agent and the cell line used in experiments 58 . rRNA degradation has also been shown to coincide with the induction of apoptosis in human lymphocytes undergoing starvation 59 and in rodent thymocytes in response to many apoptosis-inducing cellular stressors 60 . Nadano et al . demonstrated that activation of the Fas receptor with an anti-Fas antibody also induced both rRNA degradation and apoptosis in human U937 myeloid leukemia cells treated with tumour necrosis factor alpha 61 . However, in one of these studies 60 , rRNA degradation was found to occur in a caspase/Bcl-2-independent manner, suggesting that apoptotic pathways may not be required for rRNA degradation to occur. In our studies, though we observed RNA disruption reproducibly when K562 cells were treated with NK cells from different healthy volunteers, we found important variation in the degree of RNA disruption occuring in treated K562 cells. This variation may be due to several factors. Our donors were from both sexes and of varying age. NK cell activity has been found to vary by sex, with men having greater NK cytotoxicity than women 62 – 64 . In addition, NK cell cytotoxicity also varies with age, with older persons having lower cytoxocity than younger persons 65 , 66 . Other factors also appear to affect NK cell cytotoxicity, including obesity 67 and smoking 68 , 69 . Only non-smokers were invited to provide peripheral blood for this study. Pre-activation of NK cells with IL-2 augments NK cell-mediated RNA disruption of K562 cells. It has been shown that long-term incubation of NK cells with IL-2 (up to 6 days) enhances NK cell cytotoxicity towards target cell lines, although the same effect can be observed over shorter-term incubations of up to 24 h 70 , 71 . Moreover, similar to our study, Somanchi et al. 54 observed (using image cytometry) that primary human NK cells were cytotoxic to K562 cells after 4 h of incubation (although at an NK:K562 ratio of 10:1). The authors also observed that pre-stimulation of the NK cells with 50 IU mL − 1 IL-2 resulted in complete lysis of K562 cells after 4 h at an NK:K562 ratio of only 2:1 54 . Zamai et al. used both flow cytometry and a 51 Cr-release assay to demonstrate significantly greater NK cell-mediated cytotoxicity against K562 cells when NK cells were pre-incubated overnight with 100 IU mL − 1 IL-2 compared to unstimulated NK cells 72 . Clinically, the combination of IL-2 with ipilimumab, an inhibitor of the inhibitory T cell receptor CTLA-1, has been shown to increase survival in patients with metastatic melanoma compared to ipilimumab on its own 73 . Furthermore, Lotzova et al . demonstrated that the NK cell impairment characteristic of leukemia patients can be reversed in culture with IL-2, and that fully cytotoxic NK cells can be maintained and expanded in vitro 74 . What is the mechanism by which IL-2 improves NK cell cytotoxicity? Lehmann et al. found that pre-activation of primary human NK cells with IL-2 resulted in increased K562 and ML-2 leukemia cell death due to improved binding of perforin to tumour cell membranes and subsequent lysis of tumour cells 75 . This perforin-dependent mechanism for potentiation of NK cell cytotoxicity by IL-2 was also observed in another study using K562 target cells, where in vitro pre-incubation of NK cells with IL-2 for 6 and 24 h corrected for a perforin deficiency that promoted reduced NK cell efficacy in people over the age of 70 76 . Association of NK cell-mediated RNA disruption with NK cell-induced cell death in K562 cells. We found that NK cell-dependent RNA disruption in K562 cells was associated with cell death, as defined by an increase in the percentage of K562 cells with a non-viable subG1 DNA content (Fig. 4 , Supplementary Fig. S6). These findings are consistent with our previously published works showing that chemotherapeutic drug-dependent RNA disruption is associated with the onset of cell death 11 , 16 . Moreover, unlike the clonogenic, trypan blue exclusion and cell counting kit-8 assays, which respond to low, sub-lethal concentrations of drugs that typically only induce cell cycle arrest, the RDA responds solely to drug concentrations that cause cell destruction (as defined by reduced cell numbers) 16 . This may explain why high breast tumour RNA disruption during neoadjuvant chemotherapy is associated with complete tumour destruction (pCR) and improved disease-free survival after treatment 17 . The RDA is currently being assessed in a multi-institutional international clinical trial (BREVITY) for its ability to predict outcome from neoadjuvant chemotherapy in patients with breast cancer ( https://clinicaltrials.gov/ct2/show/ NCT03524430). Doxorubicin enhances NK cell-mediated RNA disruption and cell death in K562 cells. Previous investigations clearly show that treating tumour cells with low doses of chemotherapy agents can augment immune cell killing. For example, Borrelli et al. observed that treating multiple myeloma cells with sub-lethal doses of doxorubicin and melphalan (an alkylating agent) led to senescence in these cells, causing increased expression of IL-15 and NK cell proliferation. Direct or exosome-mediated IL-15 trans-presentation to NK cells resulted in increased NK cell activation and proliferation, and thus, enhanced NK cell-tumour immune surveillance 77 . Soriani et al . also observed that mouse melanoma cells treated with low doses of doxorubicin, melphalan and bortezomib exhibited a stress-induced senescent phenotype that stimulated the expression of NKG2D and DNAM-1 on their cell surface, which are well known NK cell-activating ligands 78 . This was further supported by an additional study in a mouse melanoma model where low doses of melphalan promoted the establishment of a senescent tumour cell population with higher cell surface expression of NKG2D and DNAM-1, and greater recognition by NK cells in vivo 79 . A similar phenomenon was observed with the chemotherapy agent 5-fluorouracil, which increased the expression of MHC I and NKG2D on Panc02 pancreatic cancer cells in mice. NK cells isolated from these mice exhibited enhanced cytotoxicity against Panc02 cells 80 . Chemotherapy agents have also been shown to augment NK cell-mediated tumour cell death by promoting expression of death receptors on the surface of tumour cells. Wennerberg et al. demonstrated that pre-treatment of various human tumour cell lines for 16 h with low doses of doxorubicin (184–920 nM) resulted in a 2.5-fold increase in NK cell-mediated tumour cell lysis through increased apoptosis-inducing TRAIL receptor signaling. This was further confirmed in a xenogeneic tumour-bearing mouse model, where NK cell-dependent delays in tumour progression were further prolonged in mice pre-treated with doxorubicin compared to untreated control mice 81 . Given the close association between RNA disruption and cell death 11 , 16 , it is therefore not surprising that pre-incubation of K562 cells with doxorubicin in our study augmented NK cell-mediated RNA disruption. NK cells and doxorubicin induce similar RNA disruption patterns. Interestingly, NK cell-dependent changes to the RNA banding profile of K562 cells are qualitatively very similar to those resulting from doxorubicin treatment (Fig. 6 ). This suggests that despite the contrasting mechanisms by which doxorubicin and NK cells promote the death of tumour cells, they both ultimately induce RNA disruption. Taken together with our previously published findings that different chemotherapy drugs and cellular stressors induce RNA disruption in vitro 11 , 16 and/or in vivo 14 , 17 , 82 , our work here suggests that RNA disruption may be a hallmark feature of dying tumour cells. However, the time required to evoke substantial RNA disruption appears to be much shorter for NK cells (4 h in this study) than for doxorubicin (48–72 h, depending upon the drug dose) 11 . How do various chemotherapy drugs, cellular stressors and NK cells all trigger RNA disruption? One possibility is the ability of chemotherapy agents 83 – 85 , cellular stressors 86 , 87 and granzymes released by NK cells 88 to generate reactive oxygen species (ROS) in tumour cells. These ROS can strongly reduce ribosome catalytic activity 89 and promote a variety of mutations in rRNAs, including guanine base oxidation, strand scission and rRNA-protein cross-links (recently reviewed by Shcherbik and Pestov 90 , and Tanaka and Chock 91 ). ROS generation can then promote RNA disruption by causing mutations in rRNAs, which render them non-functional and activate the 28S/18S non-functional RNA decay 92 and ribophagy 93 pathways. In addition, the generation of ROS can promote cell death by oxidizing permeability transition pore channels within mitochondria, resulting in the release of a variety of pro-apoptotic factors 94 . Protein oxidation within the endoplasmic reticulum can induce the unfolded protein response 95 , which if left unchecked, activates pro-apoptotic pathways 96 . Whereas transient activation of the non-functional rRNA decay and ribophagy pathways may permit cells to survive exposure to cellular stressors and chemotherapy agents by reducing the energetically costly process of protein translation, prolonged activation of these pathways appears to promote cell death through organelle destruction and the activation of cell death pathways. Possible use of the RDA to monitor response to immune checkpoint inhibitory drugs. Recent evidence suggests that the addition of immune checkpoint inhibitors to cytotoxic chemotherapy regimens strongly improves treatment outcome for patients with a variety of cancers. For example, the combination of the PD1 inhibitor pembrolizumab with chemotherapeutic drugs carboplatin and paclitaxel has now been adopted as first-line therapy in patients with metastatic squamous non-small cell lung carcinoma 97 . Moreover, the combination of pembrolizumab with doxorubicin and cyclophosphamide has been shown to significantly improve pCR rates in patients with high-risk breast cancer relative to standard neoadjuvant chemotherapy alone 28 . Given that NK cells have been shown in this study to induce RNA disruption in K562 tumour cells (which can be augmented by pre-treatment of the K562 cells with doxorubicin), and that this RNA disruption is reproducibly associated with the onset of tumour cell death, we are now assessing whether the RDA can also be used to predict patient response and outcome from treatments involving immune checkpoint inhibitory drugs (alone or in combination with cytotoxic chemotherapy drugs). Unlike this study, where the amount of RNA isolated from K562 cells at very high doxorubicin doses or high NK:K562 co-culture ratios precluded our ability to compute RDI values, we have observed in the BREVITY clinical trial that tumour RNA yields from on-treatment biopsies of breast cancer patients appear to be sufficiently high in the vast majority of cases to obtain RDI values for treatment outcome prediction (manuscript in preparation). In summary, we have observed in this study that K562 chronic myeloid leukemia cells treated with NK cells exhibit high RNA disruption (Fig. 1 ), a loss of plasma membrane integrity (Fig. 3 ) and increased subG1 DNA content (Fig. 4 , Supplementary Fig. S6), suggesting a strong positive association between RNA disruption, immunocytotoxicity and cell death in NK cell-treated K562 cells. Pre-incubation of K562 cells with doxorubicin augmented NK cell-induced RNA disruption (Figs. 5 a and b) and NK cell-induced reductions in RNA concentration in K562 cells (Fig. 5 c). One of the previously recognized advantages of the RDI as a predictive biomarker when administering chemotherapy drugs is that it can predict complete tumour cell destruction post-treatment (pCR) when measured after only one cycle of chemotherapy 82 . Our findings in this study raise the prospect that the RDA may be similarly useful in predicting early in treatment patient outcome from immunotherapies, possibly with or without co-administration of chemotherapy drugs. Methods Cell culture. The non-adherent K562 human chronic myeloid leukemia cell line was selected for this investigation since it is sensitive to NK cell-mediated killing due to very low expression of MHC class I 98 . It was purchased from the American Type Culture Collection. K562 cells were maintained in IMDM medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Gibco). K562 cells were cultured in 75-cm 2 vented flasks for suspension cells (Sarstedt) in a humidified, water-jacketed incubator set to 37°C and 5% CO2. Cells were allowed to proliferate to a maximum density of 1 million cells mL − 1 prior to subculture (every 3–4 days). K562 cultures were confirmed to be free of Mycoplasma infection by performing polymerase chain reaction experiments using a Mycoplasma detection kit from Applied Biological Materials. Recruitment of blood donors for isolation of NK cells. Blood donors were recruited following the study protocol approved by Laurentian University’s Research Ethics Board (no. 6016116) and the Health Sciences North Research Ethics committee. The studies involving human volunteers were performed in accordance with the Canadian TriCouncil policies on ethical conduct for research involving humans (TCPS2 2022). Each donor provided written informed consent prior to participation in this study. Healthy volunteers were recruited from Sudbury, ON, Canada through advertisements to staff and students associated with Laurentian University, Health Sciences North and the Northern Ontario School of Medicine. Inclusion criteria for donors were: persons between 20–59 years old, weight over 54 kg, non-smoker, no prior history of anemia, no chronic inflammatory diseases, and no regular use of anti-inflammatory drugs. A registered phlebotomist collected 50–60 mL of blood from each donor once a week for up to 5 weeks. Venous whole blood from each volunteer was collected in seven 10-mL Vacutainer heparinized blood collection tubes (BD), and tubes were inverted several times to ensure proper mixing. Eleven donors provided whole blood for this study. Samples from 2 donors were used for optimization of experimental procedures, after which blood cells from 9 donors were used for the research study. Multiple replicate experiments involving a minimum of 3 independent donors were performed to obtain the data depicted in this investigation. The protocols used for both blood collection and for the performance of all described experiments were approved by Health Sciences North’s Biohazard Safety Committee. Isolation of human peripheral blood mononuclear cells (PBMCs) . PBMCs were isolated from human whole blood using SepMate-50 tubes (Stemcell Technologies) following the manufacturer’s guidelines. The density gradient medium used was Ficoll-Paque PLUS density gradient media (GE Healthcare Life Sciences), and the wash and resuspension buffers consisted of EasySep Buffer (Stemcell Technologies). Platelets were removed from the recovered PBMC preparation as follows. PBMCs were centrifuged at 200 × g for 10 min followed by a slow deceleration. After removal of the supernatant, the PBMC pellet was resuspended in 1 mL of EasySep Buffer. Cells were then counted using a haemocytometer. Enrichment of NK cells from the PBMC population . Typically, NK cells account for only 5–20% of cells within the PBMC population 35 . NK cells were thus enriched within the cell mixture using the EasySep™ Direct Human NK Cell Isolation Kit (Stemcell Technologies) with the EasySep Magnet (Stemcell Technologies) following the manufacturer’s guidelines. The recommended medium (EasySep Buffer) was used. The recovered enriched NK cells were pelleted by centrifugation at 400 × g for 3 min and resuspended in 1 mL of IMDM medium supplemented with 10% FBS. Cells were then counted using a haemocytometer. Assessment of purity of NK cell preparations by flow cytometry. Flow cytometry was performed to confirm that the purity of NK cells after enrichment from the PBMC population was > 80%. Because NK cells have the surface cell differentiation markers CD45 and CD56, but lack the surface cell differentiation marker CD3 99 , we used the following fluorochrome-conjugated antibody clones, all obtained from BD Biosciences, to calculate the percentage of cells with a CD56 + CD3- CD45 + expression profile: (i) PE-CF594 mouse anti-human CD45 (clone HI30), (ii) PE-Cy7 mouse anti-human CD56 (NCAM-1) (clone B159), and (iii) BB515 mouse anti-human CD3 (clone UCHT1). eBioscience Fixable Viability Dye (FVD) eFluor 780 (Invitrogen) was also included to irreversibly label dead cells. No staining, FVD staining, single-antibody staining and fluorescence-minus-one controls (where one antibody is removed at a time to reveal the amount of fluorescent spillover of other fluors in the panel into the left-out parameter) were used to determine gates and compensation for flow cytometric characterization of both PBMCs and enriched NK cells. PBMCs and enriched NK cells were collected by centrifugation at 400–500 × g for 5 min, and resuspended in 1 × phosphate-buffered saline solution (PBS) without Ca 2+ and Mg 2+ (Cytiva, Hyclone) supplemented with (immunofluorescence staining) or without (FVD staining) 1% bovine serum albumin (BSA) to a final concentration of 10 million cells mL − 1 (immunofluorescence staining) or 1 million cells mL − 1 (FVD staining). Then, 1 µL of FVD or 50 µL of fluorochrome-conjugated antibody were added per mL of cell suspension. Mixtures were incubated in the dark for 15–30 min at 4°C. Cells were then collected by centrifugation and washed with PBS containing 1% BSA. Cells were again pelleted and resuspended to a final concentration of 1 million cells mL − 1 in cold (4°C) PBS containing 1% BSA. Then, 200 µL of each sample were transferred to a 96-well tissue culture-treated microplate (Corning) and analyzed using the CytoFLEX LX flow cytometer with the CytExpert software (Beckman Coulter). A total of 10,000 events were measured per sample. NK cells were successfully enriched to at least 90% purity (Supplementary Fig. S1 ). Activation of NK cells with IL-2 . NK cells freshly isolated from PBMCs were first resuspended in RPMI-1640 medium (HyClone) supplemented with 10% FBS. Immediately, NK cells were plated in 24-well tissue culture-treated flat-bottom plates (Sarstedt) at a density of 1 million cells per well in 1 mL of growth medium supplemented with or without 100 or 1,000 IU mL - 1 recombinant human IL-2 (Peprotech). IL-2 concentrations were selected based on the most common doses used in prior experiments with NK cells 72 , 100 , 101 . Cultures were incubated at 37°C overnight (approximately 16 h). Cultures were then centrifuged at 400 × g for 3 min, and the cell pellets were resuspended in IMDM medium supplemented with 10% FBS to a final density of 4 million cells mL - 1 . Cells were then used to treat K562 cells as described below. Pre-treatment of K562 cells with doxorubicin . Doxorubicin was kindly provided by the pharmacy at Health Sciences North (Sudbury, ON, Canada). K562 cells were plated in 6-well tissue culture-treated plates (Sarstedt) at a density of 250,000 cells per well in 3 mL of growth medium supplemented with 0, 1 or 10 µM doxorubicin. Suspension cultures were then incubated as described above for 48 h. Incubation of NK cells with K562 tumour cells. K562 suspension cultures, pre-treated with or without doxorubicin, were centrifuged at 500 × g for 5 min. The culture supernatant was discarded, and pelleted cells were resuspended in fresh drug-free IMDM medium supplemented with 10% FBS. One hundred microliters of K562 cell suspension (containing 100,000 cells) were mixed with 100 µL of unstimulated or IL-2-activated NK cell suspension (containing 50,000 to 400,000 cells to obtain NK:K562 ratios of 0.5:1, 1:1, 2:1 or 4:1) in a 96-well flat-bottom tissue culture-treated plate (Corning). “K562 cells only” and “NK cells only” controls were prepared in parallel. “K562 cells only” controls comprised 200 µL of K562 cell suspension (containing 100,000 cells) devoid of NK cells, whereas “NK cells only” controls consisted of 200 µL of NK cell suspension (containing 50,000 to 400,000 cells) lacking K562 cells. Cell mixtures were incubated at 37°C for 0 or 4 h. Removal of NK cells from K562 cells after incubation. When appropriate, the NK cells were removed from the NK/K562 co-cultures by a CD56 + selection method using the EasySep Human CD56 Positive Selection Kit II (Stemcell Technologies) with the EasySep Magnet (Stemcell Technologies). Subsets of the “K562 cells only” and “NK cells only” controls were also submitted to CD56 + selection using this method. Briefly, cultures were centrifuged at 500 × g for 5 min. The supernatants were removed and discarded, and the cell pellets were re-suspended in 100 µL of EasySep Buffer. Removal of the NK cells proceeded following the manufacturer’s instructions, with the following exceptions. The final supernatants (containing intact and/or lysed CD56-negative K562 cells) were recovered; the NK cells remaining in the tube (due to their binding to CD56-positive magnetic beads that adhered to an external magnet) were discarded. The recommended medium (EasySep Buffer) was used. The supernatant was then centrifuged at 500 × g for 5 min, and the pelleted cells were used for either DNA content analysis by flow cytometry or RNA integrity assessement, as described below. Immunocytotoxicity assays. The ability of NK cells to induce K562 cell cytotoxicity was assessed by monitoring the loss of plasma membrane integrity in K562 cells using the Incucyte S3 Live-Cell Analysis System (Sartorius) with the Incucyte Cytolight Rapid Red Dye (Sartorius) and the Incucyte Cytotox Green Dye (Sartorius) following a protocol adapted from the manufacturer’s guidelines. Briefly, K562 cultures were centrifuged at 500 × g for 5 min, and the resulting cell pellets were re-suspended and washed in 10 mL of PBS. Cells were collected by centrifugation as above and re-suspended in sufficient PBS to achieve a cell density of 1 million cells mL - 1 . To label live K562 cells, the cell-permeable, red-fluorescing Incucyte Cytolight Rapid Red Dye was added to the cell suspensions at a final concentration of 3 µM. The mixtures were incubated at 37ºC for 20 min with periodic mixing. Excess dye was then removed by adding 6-fold excess volume of IMDM containing 10% FBS and harvesting the cells by centrifugation as above. Pelleted Rapid Red Dye-labelled cells were re-suspended in IMDM supplemented with 10% FBS. Then, 50 µL of K562 cell suspension (containing 50,000 cells) and 50 µL of NK cell suspension (containing 25,000-200,000 cells) were plated in 96-well flat-bottom plates (Corning). For “K562 cells only” and “NK cells only” controls, 100 µL of the appropriate cell suspension (containing an equivalent number of cells) were plated. After plating cells, Incucyte Cytotox Green Dye, a cell-impermeable, green-fluorescing dye used to label cells with compromised plasma membranes, was added directly to wells to yield a final dye concentration of 250 nM. Cells were permitted to settle to the bottom of the plate for 20–30 min prior to imaging. Plates were then placed in the Incucyte S3 Live-Cell Analysis System at 37°C and 5% CO 2 for imaging at 10 × magnification and counting using Incucyte’s 2018C software every 15 min for 4 h. Labelled NK cells were excluded from K562 cell counts using the instrument’s size filter feature, as NK cells are considerably smaller than K562 cells (Supplementary Figs. S2 and S4). DNA content analyses by flow cytometry. The ability of NK cells to induce K562 cell death was assessed by quantifying K562 cells with a subG1 DNA content using a flow cytometry procedure adapted from Butler et al . 11 . Briefly, 50,000 K562 cells with or without (“K562 cells only” controls) 25,000-200,000 NK cells were cultured for 0, 4, 24 and 48 h in 800 µL of IMDM supplemented with 10% FBS in 24-well flat-bottom plates (Sarstedt). Cultures were centrifuged at 500 × g for 5 min. Pelleted cells were then resuspended and washed twice with 2 mL of PBS, and collected by centrifugation at 233 × g for 10 min. Next, cell pellets were re-suspended in 0.5 mL of PBS and placed on ice. Cells were immediately fixed by mixing 1.5 mL of cold (-20°C) anhydrous ethanol with the cell suspensions. Fixed cells were stored at -20°C until further use. Fixed cell suspensions were centrifuged at 530 × g for 10 min. Cell pellets were resuspended and washed with 2 mL PBS, and again harvested by centrifugation. Cells were stained by re-suspending the pellets with 500 µL of propidium iodide staining solution (100 µg mL - 1 propidium iodide, 100 µg mL - 1 RNase A, 0.3% NP-40, and 0.3% sodium citrate) and incubating the suspensions at 37°C for 30 min. Stained cells were then analyzed using a Cytomics FC500 flow cytometer (Beckman Coulter) with the 675-nm bandpass filter (488-nm excitation wavelength). Samples were measured for 20,000 events or 300 sec, whichever occurred first. Data analyses were performed using the CXP Analysis software (Beckman Coulter) without the use of gating. RNA disruption assay . K562 cells were collected from cultures by centrifugation at 500 × g for 5 min. Total RNA was then isolated from pelleted cells using the RNeasy Mini kit (Qiagen) following the manufacturer’s protocol titled Purification of Total RNA from Animal Cells using Spin Technology. Cells were homogenized using a needle, as described by the manufacturer. RNA concentrations were measured using the NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific). Total RNA was resolved by capillary gel electrophoresis using the 2100 Bioanalyzer (Agilent Technologies) with the RNA 6000 Nano kit (Agilent Technologies) per the manufacturer’s instructions. RNA electropherogram data files were transmitted electronically to Rna Diagnostics for RDI calculation. The RNA electropherograms depicted in Figs. 2 a, 5 a, and 6 a are composites of individual experiments conducted using 12 well RNA Nanochips ™ and are representative of three independent experiments. Graphics. Plots were prepared using Prism. Electropherograms were generated using the 2100 Expert software version B.02.09.SI725 (SR1) (Agilent Technologies). Flow cytometry plots for purity assessment of NK cell preparations and DNA content analyses experiments were produced using the CytExpert software (Beckman Coulter) and the CXP Analysis software (Beckman Coulter), respectively. Declarations Acknowledgements The authors would like to thank Drs. Carita Lanner and Tom Kovala (NOSM University and Laurentian University, Sudbury, ON, Canada), as well as Dr. Hoang-Thanh Le (Health Sciences North Research Institute, Sudbury, ON, Canada) for their input into the design of studies, the interpretation of experiments, and/or critical feedback on the manuscript. They would also also like to thank Dr. Graham Pawelec (University of Tübingen, Tübingen, Germany) for providing guidance on the design of immunocytotoxicity experiments and Renée St. Onge for her throughout editorial review of the manuscript prior to submission. This work was supported by a MITACS Accelerate graduate student internship award (IT2731) to I.P. and a grant from the Northern Cancer Foundation (Sudbury, ON, Canada) to A.P., along with in-kind support from Rna Diagnostics (Sudbury and Toronto, ON, Canada). Author contributions A.M.P. and A.K. raised funds and played a role in hypothesis generation and the design of experiments. A.K. served as the academic supervisor for this MITACS-funded internship project. I.P. and A.M.P. wrote the manuscript, and all authors participated in its review. I.P. conducted all of the experiments and prepared the manuscript figures. B.G. advised I.P. on experimental protocols and use of the live-cell imaging system and flow cytometer. L.B.P. computed RDI values for all samples. All authors reviewed the manuscript. Competing interests Members of the team disclose the following significant conflicts of interest. A.M.P. holds a large number of shares in Rna Diagnostics, Inc., a company that is commercializing the RDA for clinical use. He also serves as the company’s Chief Scientific Officer. L.B.P. is Vice President of Research and Development for Rna Diagnostics. A large portion of B.G.’s salary and benefits was supported by Rna Diagnostics during this investigation. A.M.P. and B.G. have and will receive royalties from the commercialization of the RDA. I.P and A.K. have no competing interests to disclose. Availability of data and materials Supplementary figures, and electropherogram files used to create manuscript figures can be found in the supplementary information pdf file ESM_1. 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Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry. Cytometry 32, 280–285 (1998). Prieto, P.A., et al. CTLA-4 blockade with ipilimumab: long-term follow-up of 177 patients with metastatic melanoma. Clin Cancer Res 18, 2039–2047 (2012). Lotzova, E., Savary, C.A. & Herberman, R.B. Induction of NK cell activity against fresh human leukemia in culture with interleukin 2. J Immunol 138, 2718–2727 (1987). Lehmann, C., Zeis, M. & Uharek, L. Activation of natural killer cells with interleukin 2 (IL-2) and IL-12 increases perforin binding and subsequent lysis of tumour cells. Br J Haematol 114, 660–665 (2001). Rukavina, D., et al. Age-related decline of perforin expression in human cytotoxic T lymphocytes and natural killer cells. Blood 92, 2410–2420 (1998). Borrelli, C., et al. Drug-Induced Senescent Multiple Myeloma Cells Elicit NK Cell Proliferation by Direct or Exosome-Mediated IL15 Trans-Presentation. Cancer Immunol Res 6, 860–869 (2018). 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J Natl Cancer Inst 108, 1–2 (2016). Mizutani, H., Tada-Oikawa, S., Hiraku, Y., Kojima, M. & Kawanishi, S. Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide. Life Sci 76, 1439–1453 (2005). Conklin, K.A. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr Cancer Ther 3, 294–300 (2004). Yang, H., et al. The role of cellular reactive oxygen species in cancer chemotherapy. J Exp Clin Cancer Res 37, 266 (2018). Scherz-Shouval, R., et al. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J 26, 1749–1760 (2007). Tominaga, H., Kodama, S., Matsuda, N., Suzuki, K. & Watanabe, M. Involvement of reactive oxygen species (ROS) in the induction of genetic instability by radiation. J Radiat Res 45, 181–188 (2004). Prager, I. & Watzl, C. Mechanisms of natural killer cell-mediated cellular cytotoxicity. J Leukoc Biol 105, 1319–1329 (2019). Willi, J., et al. Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center. Nucleic Acids Res 46, 1945–1957 (2018). Shcherbik, N. & Pestov, D.G. The impact of oxidative stress on ribosomes: from injury to regulation. Cells 8, 1–16 (2019). Tanaka, M. & Chock, P.B. Oxidative modifications of RNA and its potential roles in biosystems. Front Mol Biosci 8, 685331 (2021). LaRiviere, F.J., Cole, S.E., Ferullo, D.J. & Moore, M.J. A late-acting quality control process for mature eukaryotic rRNAs. Mol Cell 24, 619–626 (2006). Kraft, C., Deplazes, A., Sohrmann, M. & Peter, M. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat Cell Biol 10, 602–610 (2008). England, K. & Cotter, T.G. Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis. Redox Rep 10, 237–245 (2005). Zhang, Z., et al. Redox signaling and unfolded protein response coordinate cell fate decisions under ER stress. Redox Biol 25, 101047 (2019). Fribley, A., Zhang, K. & Kaufman, R.J. Regulation of apoptosis by the unfolded protein response. Methods Mol Biol 559, 191–204 (2009). Paz-Ares, L., et al. Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer. N Engl J Med 379, 2040–2051 (2018). Martin, B.K., et al. Induction of MHC class I expression by the MHC class II transactivator CIITA. Immunity 6, 591–600 (1997). Pascal, V., et al. Comparative analysis of NK cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditions. Eur J Immunol 34, 2930–2940 (2004). Ghasemi, R., et al. Selective targeting of IL-2 to NKG2D bearing cells for improved immunotherapy. Nat Commun 7, 12878 (2016). Sanchez-Martinez, D., et al. Expansion of allogeneic NK cells with efficient antibody-dependent cell cytotoxicity against multiple tumors. Theranostics 8, 3856–3869 (2018). Additional Declarations Competing interest reported. Members of the team disclose the following significant conflicts of interest. A.M.P. holds a large number of shares in Rna Diagnostics, Inc., a company that is commercializing the RDA for clinical use. He also serves as the company’s Chief Scientific Officer. L.B.P. is Vice President of Research and Development for Rna Diagnostics. A large portion of B.G.’s salary and benefits was supported by Rna Diagnostics during this investigation. A.M.P. and B.G. have and will receive royalties from the commercialization of the RDA. I.P and A.K. have no competing interests to disclose. Supplementary Files ESM1.pdf ESM2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2944450","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":205891572,"identity":"07f314fc-43e7-4572-9609-a59fe28d6da0","order_by":0,"name":"Isabella Pascheto","email":"","orcid":"","institution":"Laurentian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Isabella","middleName":"","lastName":"Pascheto","suffix":""},{"id":205891573,"identity":"3f12ece6-966c-4da1-9b46-a0507063ddb9","order_by":1,"name":"Baoqing Guo","email":"","orcid":"","institution":"Health Sciences North Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Baoqing","middleName":"","lastName":"Guo","suffix":""},{"id":205891574,"identity":"57252095-b67a-4e00-bae9-33ba830a6356","order_by":2,"name":"Aseem Kumar","email":"","orcid":"","institution":"Laurentian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aseem","middleName":"","lastName":"Kumar","suffix":""},{"id":205891575,"identity":"dcadc53a-7e7d-4876-846b-19d065c11f4c","order_by":3,"name":"Laura B. Pritzker","email":"","orcid":"","institution":"Rna Diagnostics, Inc., Sudbury and Toronto","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Laura","middleName":"B.","lastName":"Pritzker","suffix":""},{"id":205891576,"identity":"afb8bc8e-e121-4f9c-b30b-25ba918bae5a","order_by":4,"name":"Amadeo Mark Parissenti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYLCCBDDJ2PgYRLERr4WNsdmYeC0MEKVs0kQp5J/d/OzDgz91cubyzW3VBX/u5PMxMD/8gE+LxJ1jxjMS2w4bW7Yxtt2e2fbMso2BzVgCrzU3EowZEhsOJG44BtTC23DYgI2BhwGvFvkb6Z8ZEv7U1YO0FPP8AWth/oFPi8GNHGOGBDbmBAOgFmYeNrAWNry2GN45U8wA9IvhhmOJzdK8bUAtzGxmFvi0yN1u38z440+dvMHh4w8/gxwm3978+AY+LVh8yoxXPVYto2AUjIJRMArQAACM0UXWO4MJ9gAAAABJRU5ErkJggg==","orcid":"","institution":"Laurentian University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Amadeo","middleName":"Mark","lastName":"Parissenti","suffix":""}],"badges":[],"createdAt":"2023-05-16 21:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2944450/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2944450/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":38028166,"identity":"3510a858-1f23-49e3-a346-0bbe567c623a","added_by":"auto","created_at":"2023-06-05 14:37:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":523262,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NK cells on RNA disruption and RNA abundance in K562 cells. K562 cells were incubated with or without NK cells at an NK:K562 ratio of 0.5:1, 1:1, 2:1 and 4:1. After 0 or 4 h of incubation, total RNA was isolated from K562 cells. When appropriate, NK cells were removed from cultures prior to RNA extractions using CD56+ selection. (\u003cstrong\u003ea\u003c/strong\u003e) RNA was size-separated by capillary gel electrophoresis. Arrows indicate the location of full-length 28S and 18S rRNAs. The composite electropherogram is representative of four independent biological replicate experiments. Lanes 1 to 6 are from electropherogram 17-55-39, while lanes 7 to 12 are from electropherogram 16-28-41 (see pages 11 to 25 of ESM_1 file). (\u003cstrong\u003eb\u003c/strong\u003e) RNA disruption was quantified using the RDA. The data are presented as means ± standard errors (\u003cem\u003en = \u003c/em\u003e3-4). N/A, non-assessable sample owing to insufficient RNA. (\u003cstrong\u003ec\u003c/strong\u003e) The concentration of RNA extracts was measured using spectrophotometry. The data are presented as means ± standard errors (\u003cem\u003en =\u003c/em\u003e 4), with all samples plated in duplicate.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/4b5525fa204392e9be1726b9.png"},{"id":38028163,"identity":"f7b75d20-da1a-427f-bf21-cb736b86fa02","added_by":"auto","created_at":"2023-06-05 14:37:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":577031,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of pre-activation of NK cells with IL-2 on immune cell-mediated RNA disruption and RNA yield reduction in K562 cells. K562 cells were incubated with or without NK cells at an NK:K562 ratio of 0.5:1, 1:1 and 2:1. When appropriate, NK cells were pre-activated overnight with 0.1 or 1 IU mL\u003csup\u003e-1\u003c/sup\u003e IL-2. After 0 or 4 h of incubation, total RNA was isolated from K562 cells. When appropriate, NK cells were removed from cultures prior to RNA extractions using CD56+ selection. (\u003cstrong\u003ea\u003c/strong\u003e) RNA was size-separated by capillary gel electrophoresis. Arrows indicate the location of full-length 28S and 18S rRNAs. The composite electropherogram is representative of three independent biological replicates. Lanes 1 to 7 are from electropherogram 17-59-45, while lanes 8 to 16 are from electropherogram 18-36-48 (see pages 26 to 52 of ESM_1 file). \u0026nbsp;(\u003cstrong\u003eb\u003c/strong\u003e) RNA disruption was quantified using the RDA. The data are presented as means ± standard errors (\u003cem\u003en\u003c/em\u003e = 1-3). N/A, non-assessable sample owing to insufficient RNA. (\u003cstrong\u003ec\u003c/strong\u003e) The concentration of RNA extracts was measured using spectrophotometry. The data are presented as means ± standard errors (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/0263f551d42b6772256e4c0c.png"},{"id":38030365,"identity":"7be07391-e148-4885-99f3-fceb7e173dbd","added_by":"auto","created_at":"2023-06-05 14:45:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61470,"visible":true,"origin":"","legend":"\u003cp\u003eImmunocytotoxicity of NK cells on K562 cells as measured through loss of membrane integrity. K562 cells were co-cultured for 4 h with either unstimulated (\u003cstrong\u003ea\u003c/strong\u003e) or IL-2-activated (\u003cstrong\u003eb\u003c/strong\u003e) NK cells at an NK:K562 ratio of 0.5:1, 1:1, 2:1 and 4:1. Live K562 cells were labelled with a cell-permeable red-fluorescing dye (red cells), whereas cells with a compromised plasma membrane were labelled with a cell-impermeable green-fluorescing dye (green cells). Labelled cells were then counted using a live-cell imaging and analysis system. Data are presented as means ± standard errors (\u003cem\u003en\u003c/em\u003e = 3-4), with all samples plated in duplicate.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/188126a89e388ae09189fc92.png"},{"id":38028161,"identity":"0458d181-ce54-411d-9131-3512d2e33513","added_by":"auto","created_at":"2023-06-05 14:37:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59986,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NK cells on the DNA content of K562 cells. K562 cells were incubated for 4 h (left panel), 24 h (middle panel) or 48 h (right panel) with NK cells at an NK:K562 ratio of 0.5:1, 1:1, 2:1 and 4:1. NK cells were removed from cultures using CD56+ selection. The remaining K562 cells were then washed, fixed, stained with propidium iodide, and analysed by flow cytometry. Data are presented as means ± standard errors (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/3d9938b15370775a8ac538e7.png"},{"id":38031692,"identity":"97825c2e-c236-47cc-98b1-597212f39614","added_by":"auto","created_at":"2023-06-05 14:53:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":685508,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of doxorubicin pre-treatment on NK cell-dependent RNA disruption and RNA abundance in K562 cells. K562 cells were incubated with 0, 1 or 10 mM doxorubicin for 48 h. Cells were then collected, resuspended in fresh doxorubicin-free medium, and subsequently treated for 0 or 4 h with or without NK cells at an NK:K562 ratio of 0.5:1, 1:1 and 2:1. Total RNA was then isolated from K562 cells. When appropriate, NK cells were removed from co-cultures using CD56+ selection. (\u003cstrong\u003ea\u003c/strong\u003e) RNA was size-separated by capillary gel electrophoresis. Arrows indicate the location of full-length 28S and 18S rRNAs. The composite electropherogram is representative of three independent biological replicates. Lanes 1 to 4 are from electropherogram 21-46-14, while lanes 5 to 16 are from electropherogram 19-44-12 (see pages 53 to 73 of ESM_1 file). \u0026nbsp;(\u003cstrong\u003eb\u003c/strong\u003e) RNA disruption was quantified using the RDA. Data are presented as means ±standard errors (\u003cem\u003en\u003c/em\u003e = 2-3). N/A, non-assessable sample owing to insufficient RNA. (\u003cstrong\u003ec\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eThe concentration of RNA extracts was measured using spectrophotometry. Data are presented as means ±standard errors (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/5e46b98130b59520de835f8f.png"},{"id":38032798,"identity":"b19e962a-39aa-4192-a19f-99b76cd1c355","added_by":"auto","created_at":"2023-06-05 15:01:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":334931,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of RNA disruption patterns between doxorubicin-treated K562 cells and K562 cells co-cultured with NK cells. For doxorubicin-treated K562 cells (red labels and lines), K562 cells were treated with 10 mM doxorubicin (DOX) for 48 h (panels a and b) or 72 h (panels c and d). For NK cell-treated K562 cells (blue labels and lines), K562 cells were co-cultured for 4 h with NK cells in an NK:K562 ratio of 0.5:1 (panel a), 1:1 (panel b), 2:1 (panel c) and 4:1 (panel d). NK cells were removed from co-cultures using CD56+ selection. Total RNA was isolated from treated K562 cells and size-separated by capillary gel electrophoresis. Total RNA preparations from \u0026nbsp;doxorubicin-treated K562 cells were obtained from a previous study\u003csup\u003e11\u003c/sup\u003e, whereas total RNA preparations from NK cell-treated K562 cells were obtained from this study (Fig. 1a). \u0026nbsp;Electropherogram traces are from electropherogram 19-47-52 (see pages 74 to 83 of ESM_1 file). Peaks corresponding to full-length 28S and 18S rRNAs are labelled. FU, fluorescence units.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/de4fcfe9c2460d6c7b3915f9.png"},{"id":39138338,"identity":"bc5ad247-9ec3-46e2-aee5-c976b5b80264","added_by":"auto","created_at":"2023-06-27 02:59:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1845598,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/bdf11940-a2a7-49e8-99e8-35f158570d5c.pdf"},{"id":38028191,"identity":"0d42605e-b7f5-4614-8a8b-d13ff7c56079","added_by":"auto","created_at":"2023-06-05 14:37:45","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15992718,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/5f9e1175eb8c21cc39449ac6.pdf"},{"id":38028167,"identity":"423434bc-0725-4e75-bb91-3623f9a4dddb","added_by":"auto","created_at":"2023-06-05 14:37:43","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":74912,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2944450/v1/34a348c1700d9004d8672bcf.xlsx"}],"financialInterests":"Competing interest reported. Members of the team disclose the following significant conflicts of interest. A.M.P. holds a large number of shares in Rna Diagnostics, Inc., a company that is commercializing the RDA for clinical use. He also serves as the company’s Chief Scientific Officer. L.B.P. is Vice President of Research and Development for Rna Diagnostics. A large portion of B.G.’s salary and benefits was supported by Rna Diagnostics during this investigation. A.M.P. and B.G. have and will receive royalties from the commercialization of the RDA. I.P and A.K. have no competing interests to disclose.","formattedTitle":"Association of extensive RNA disruption with natural killer cell-mediated death of K562 chronic myelogenous leukemia cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe majority of breast cancer patients undergoing chemotherapy and/or immunotherapy treatments often suffer from toxic side effects from these treatments, including cardiotoxicity, thromboemboli, pneumonitis, hepatitis, neurotoxicities, neutropenia, and secondary cancers\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In contrast, only a minority of such patients receive a substantial survival benefit from treatment, in particular for some tumour subtypes\u003csup\u003e1,2,7\u0026minus;9\u003c/sup\u003e. Consequently, there is growing interest in treatment response assessment tools that can predict the outcome of regimens involving standard cytotoxic drugs with or without the employment of immunotherapies\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. With such tools, oncologists could determine whether their patients are responding positively during the early stages of treatment. Patients with non-responsive tumours could then forgo the costs and toxicities associated with ineffective regimens and proceed to alternate treatments.\u003c/p\u003e \u003cp\u003eA treatment response assessment tool showing promise is the RNA disruption assay (RDA). The RDA quantifies a specific phenomenon we termed \u0026lsquo;RNA disruption\u0026rsquo;, which is characterized by the degradation of the 28S and 18S ribosomal RNAs (rRNAs) with the concomitant accumulation of abnormal bands between the 28S and 18S rRNAs on RNA electropherograms. These bands are derived, at least in part, from the 28S rRNA\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In a sub-study of the NCIC-CTG MA.22\u003csup\u003e12\u003c/sup\u003e and NeoAva\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e clinical trials, we demonstrated that high mid-treatment tumour RNA disruption was associated with little to no residual disease post-treatment (as indicated by a pathologic complete response [pCR] or no residual cancer burden) and increased survival of breast cancer patients following neoadjuvant chemotherapy, suggesting that the RDA can predict treatment outcome much earlier than standard post-treatment evaluations. The assay was also able to predict response and disease-free survival after CHOP chemotherapy in canine lymphoma patients\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The predictive value of the RDA in neoadjuvant chemotherapy is currently being investigated in a large international clinical trial called \u0026lsquo;BREVITY\u0026rsquo;, involving approximately 600 female patients with advanced breast cancer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clinicaltrials.gov/ct2/show/NCT03524430\u003c/span\u003e\u003cspan address=\"https://clinicaltrials.gov/ct2/show/NCT03524430\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRNA disruption has been observed \u003cem\u003ein vitro\u003c/em\u003e in a wide variety of tumour and non-tumour cell types, including breast epithelial cells, ovarian endometrioid cells, myeloid cells, melanocytes and vascular endothelial cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. It is triggered by many structurally and mechanistically distinct chemotherapy agents as well as specific cell stressors, including endoplasmic reticulum stress, oxidative stress and nutrient/growth factor limitation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This phenomenon is associated with cell death \u003cem\u003ein vitro\u003c/em\u003e, as measured by a reduction in cell numbers, loss of cell replicative capacity, and the generation of cells with a subG1 DNA content\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Although RNA disruption was found to be associated with chemotherapy-induced tumour cell death \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and clinical response to neoadjuvant chemotherapy in cancer patients \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, its occurrence in response to an immunogenic challenge has yet to be explored.\u003c/p\u003e \u003cp\u003eImmune checkpoint inhibitors represent one of the most promising new classes of chemotherapy drugs in oncology\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These drugs strongly augment the ability of a patient\u0026rsquo;s own immune cells to recognize and destroy tumours by inhibiting their specific immunoevasion pathways used to block immune cell killing mechanisms\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. An example of an immunoevasion mechanism is the binding of programmed death ligand 1 (PDL1) on the surface of tumour cells to the programmed cell death 1 (PD1) receptor on the surface of helper T cells, which inhibits the activation of the latter cells. This blocks the production of cytokines by T cells, which would otherwise activate cytotoxic T cells. Blocking T cell activation also prevents their release of interleukin (IL)-2 and other cytokines that promote B cell proliferation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, B cell differentiation into antibody-secreting plasma cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and activation of natural killer (NK) cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Thus, PDL1 inhibitory drugs, such as pembrolizumab, can restore the ability of the immune system to recognize and kill tumours\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Interestingly, PDL1 inhibitors have resulted in dramatic improvements in the treatment of certain cancers including lung cancer\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, renal cell carcinoma\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and melanoma\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Moreover, pembrolizumab was found to strongly augment the pCR rate for breast cancer patients treated with neoadjuvant docetaxel chemotherapy in the I-SPY2 clinical trial\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Other immunomodulatory approaches, such as chimeric antigen receptor (CAR) T cell therapy, can effectively treat a variety of haematological malignancies and solid tumours. In these approaches, T cells are taken from the patient and genetically engineered to give them the ability to target specific receptors on the surface of tumour cells, such as CD19 in B cell-derived cancers and CD30 in Hodgkin lymphoma\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Unfortunately, the above immunotherapies have significant toxicities, including cytokine-release syndrome\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, neurotoxicities\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and cardiotoxicities\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (associated with CAR-T therapy), as well as pneumonitis, neuropathies, endocrinopathies, hepatitis and dermatitis (associated with PD1/PDL1 inhibitors)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs is the case with chemotherapy, only a limited number of patients respond favorably to immunotherapy. For example, \u0026lt; 20% of lung cancer patients and \u0026lt;\u0026thinsp;45% of patients with malignant melanoma respond to PDL1 inhibitors\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Similarly, recent reviews indicate that objective and complete response rates to CAR T cell therapy vary widely from study to study\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Given the large number of non-responding patients, and the costs and toxicities associated with the above immunotherapies, a biomarker that could effectively predict treatment outcome prior to treatment or early during treatment would be highly valued.\u003c/p\u003e \u003cp\u003eIn this study, we began exploring the potential utility of the RDA to quantify the response of cancer patients to immunotherapy by first determining whether immune cells can trigger RNA disruption in tumour cells \u003cem\u003ein vitro\u003c/em\u003e, and whether this disruption is accompanied by cytotoxicity and a loss of tumour cell viability. We report here that freshly isolated NK cells from healthy volunteers strongly induce RNA disruption, cytotoxicity (as measured by a reduction in plasma membrane integrity) and cell death (as measured by the generation of cells with a subG1 DNA content) in human chronic myeloid leukemia K562 cells. Moreover, pre-activation of NK cells with IL-2 or pre-treatment of K562 cells with doxorubicin augments NK cell-dependent RNA disruption in K562 cells. Taken together with our previously published findings, our work suggests that RNA disruption is strongly associated with tumour cell death, regardless of the death-inducing agent.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eFreshly isolated human NK cells induce RNA disruption in human chronic myeloid leukemia cells.\u003c/b\u003e To assess whether immune cells can induce RNA disruption in tumour cells, we first isolated peripheral blood mononuclear cells (PBMCs) from healthy human volunteers, and enriched the cell preparation for NK cells. We then treated K562 human chronic myeloid leukemia cells with freshly isolated NK cells for 4 h, and measured the extent of RNA disruption occurring in K562 cells using the RDA. To ensure that RNA from NK cells did not contribute to the signal, we removed NK cells from co-cultures prior to RNA extractions using selection on CD56\u0026thinsp;+\u0026thinsp;magnetic beads (see Methods).\u003c/p\u003e \u003cp\u003eWe found that NK cells triggered RNA disruption in K562 cells, particularly when they outnumbered their target cells (NK:K562 ratios of 2:1 and 4:1) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, compare lanes/columns 10 and 12 to lane/column 4). We noted pronounced reductions in the intensities of the 28S and 18S rRNA bands, and increased intensities of abnormal bands in the \u0026ldquo;inter-region\u0026rdquo; between the 28S and 18S rRNA bands of electropherograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). These changes in RNA banding patterns were reflected in increased RNA disruption indexes (RDIs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), which express the extent of RNA disruption as a ratio between the combined areas of the abnormal peaks and the combined areas of the intact 28S and 18S rRNA bands on RNA electropherograms\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Interestingly, we also found that the extent of RNA disruption progressively increased as NK cells became more abundant than their target cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We observed little to no RNA disruption in K562 cells when NK cells equaled or were outnumbered by K562 cells (NK:K562 ratios of 1:1 and 0.5:1, respectively) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, compare lanes/columns 6 and 8 to lane/column 4). Few bands were noted in the inter-region of electropherograms in these instances (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and relatively low RDI values were consistently calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also noted an interesting negative association between RNA disruption and RNA abundance within K562 cells. In general, as RNA disruption became more prevalent in K562 cells, RNA abundance within cells (as measured by spectrophotometry) progressively decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that NK cells also cause a reduction in RNA levels with their target cells, likely due to cell death/destruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the absence of NK cell RNA from K562 RNA preparations, we cultured NK cells in the absence of K562 cells, removed the NK cells from cultures using CD56\u0026thinsp;+\u0026thinsp;selection, and isolated and analysed total RNA from what remained. We recovered low to negligible amounts of RNA from these \u0026ldquo;NK cells only\u0026rdquo; controls (\u0026lt;\u0026thinsp;10 ng \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, columns 5, 7, 9 and 11), and noted only weak, barely detectable bands on RNA electropherograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, lanes 5, 7, 9 and 11). Furthermore, the recovered RNA was deemed \u0026lsquo;not assessable\u0026rsquo; by the RDA owing to insufficient RNA concentration to detect the 28S and 18S rRNA peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, columns 5, 7, 9 and 11). Together, these findings suggest that NK cells were effectively removed from the NK/K562 co-cultures, and that NK cell-derived total RNA did not contribute substantially to the K562 cell RNA electropherogram banding pattern or to RDI measurements.\u003c/p\u003e \u003cp\u003eBecause CD56\u0026thinsp;+\u0026thinsp;selection was required to remove NK cells from co-cultures prior to RDA analyses, we determined the impact of the selection process itself on RNA disruption in K562 cells cultured in the absence of NK cells. Although the selection process did not impact RDI values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, compare column 4 to column 3), we did note that untreated K562 cells subjected to CD56\u0026thinsp;+\u0026thinsp;selection exhibited lower-molecular-weight abnormal bands in the inter-region of RNA electropherograms than untreated K562 controls that did not undergo this selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, compare lane 4 to lane 3). We also found that the concentration of RNA isolated from selected K562 cells was lower than that of unselected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, compare column 4 to column 3), suggesting that the NK cell removal procedure likely resulted in the loss of some K562 cells, which is not unexpected with lengthy procedures. This indicates that the NK cell removal procedure had a slight effect on K562 RNA levels and banding pattern.\u003c/p\u003e \u003cp\u003eWe included 0-h controls in all of our experiments to provide a reference point as to the state of RNA disruption in K562 cells prior to treatment initiation. Untreated K562 cells (at 0 h) generally demonstrated slightly greater levels of abnormal bands in the inter-region of the RNA electropherogram and higher RDI values than untreated cells after 4 h of incubation (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, compare lane/column 2 to lane/column 4). This was likely due to stress experienced by K562 cells during processing prior to incubation, which was relieved upon incubation in high-nutrient cell culture medium containing growth factors.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePre-treatment of NK cells with IL-2 augments NK cell-mediated RNA disruption in K562 cells.\u003c/b\u003e IL-2 is a cytokine known to increase NK cell-mediated tumour cytotoxicity\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. To determine whether pre-treatment of NK cells with IL-2 could enhance their ability to induce RNA disruption in K562 cells, we repeated the above experiments using NK cells pre-treated with different concentrations of IL-2.\u003c/p\u003e \u003cp\u003eWe observed more extensive disruption in K562 cells treated with IL-2-stimulated NK cells than in K562 cells treated with unstimulated NK cells, particularly when NK cells were equally (NK:K562 ratio of 1:1) or more abundant (NK:K562 ratios of 2:1) than their target cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, compare lanes/columns 9 and 10 to lane/column 8; lanes/columns 12 and 13 to lane/column 11; and lanes/columns 15 and 16 to lane/column 14). Furthermore, we again found a negative association between RNA disruption and RNA yields, with K562 cells treated with pre-activated NK cells generally yielding less concentrated RNA extracts than K562 cells treated with unstimulated NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, compare columns 9 and 10 to column 8; columns 12 and 13 to column 11; and columns 15 and 16 to column 14). Taken together, our findings suggest that pre-activation of NK cells with IL-2 enhances NK cell-mediated RNA disruption and reduces total RNA content in K562 cells in a dose-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe confirmed the successful removal of pre-activated NK cells from co-cultures prior to RNA isolation from K562 cells as above. Here, we tested only the highest density NK cell mono-culture (corresponding to cell amounts found in co-cultures with an NK:K562 ratio of 2:1), pre-treated or not with IL-2. We reasoned that if the largest number of NK cells used to treat K562 cells was effectively removed during CD56\u0026thinsp;+\u0026thinsp;positive selection, then the smaller amounts of NK cells in the co-cultures with lower NK:K562 ratios would also have been successfully removed. We recovered little to no RNA from the \u0026ldquo;NK cells only\u0026rdquo; controls (\u0026lt;\u0026thinsp;10 ng \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), irrespective of the IL-2 dose used to pre-activate NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, columns 5, 6 and 7), and what was recovered was deemed un-assessable by the RDA owing to insufficient RNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, columns 5, 6 and 7). Overall, these controls revealed that NK cells were effectively removed from co-cultures, and there was little contribution of NK cell RNA towards electropherograms of RNA isolated from purified K562 cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNK cells pre-incubated with or without IL-2 induce loss of membrane integrity in K562 tumour cells.\u003c/b\u003e An earlier study found that freshly isolated NK cells are cytotoxic to K562 cells\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. To explore this further, we co-cultured K562 cells with unstimulated NK cells for 4 h, and monitored the loss of plasma membrane integrity using a live-cell imaging and analysis system with a live/dead cell-labelling assay. As the plasma membrane is responsible for protecting the cell from its surroundings and for transporting essential substances in and out of the cell, loss of its integrity often precedes cell death\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, loss of plasma membrane integrity is commonly used as a marker of immunocytotoxicity\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe found that the number of K562 cells with a compromised plasma membrane (normalized to total live cells) increased when K562 cells were co-cultured with NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, compare columns 3\u0026ndash;6 to column 2), suggesting that NK cells trigger a loss of membrane integrity in K562 cells. Furthermore, the number of K562 cells exhibiting loss of membrane integrity increased with increasing numbers of co-incubated NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the effect of IL-2 pre-activation on NK cell-mediated loss of K562 cell membrane integrity, we repeated our experiments using NK cells pre-treated overnight with IL-2. We detected a greater number of membrane-compromised K562 cells in co-cultures containing IL-2-stimulated NK cells than in co-cultures containing unstimulated NK cells, with numbers increasing with increasing IL-2 doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, compare columns 4 and 5 to column 3; columns 7 and 8 to column 6; and columns 10 and 11 to column 9), suggesting that IL-2 activation promotes NK cell-dependent cytotoxicity towards K562 cells.\u003c/p\u003e \u003cp\u003eWhile conducting these experiments, the imaging system\u0026rsquo;s parameters were adjusted to exclude counting of labelled NK cells. To ensure that labelled NK cells in co-cultures were not counted in error by the imaging system, we performed the live/dead cell-labelling assay using NK cells (with or without prior IL-2 activation) cultivated in the absence of K562 cells. We found that the amount of labelled NK cells counted was negligible (Supplementary Figs. S2-S5). These findings confirm that NK cells were effectively filtered out by the imaging system, and that counts of membrane-compromised cells in co-cultures stemmed exclusively from K562 cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNK cells promote the generation of non-viable K562 tumour cells with a subG1 DNA content.\u003c/b\u003e Cells with a subG1 DNA content are non-viable as they lack sufficient genetic material to maintain cellular processes\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, thus making the increased detection of cells with a subG1 DNA content a robust marker of cell death. Given NK cells\u0026rsquo; impact on the plasma membrane of K562 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we next assessed the ability of NK cells to increase the abundance of K562 cells with a subG1 DNA content. We co-cultured NK cells with K562 cells at different NK:K562 ratios for 4, 24 and 48 h. After removing the NK cells from co-cultures, K562 cells were fixed and stained with propidium iodine. The percentage of K562 cells with a subG1 DNA content was then determined using flow cytometry.\u003c/p\u003e \u003cp\u003eWe found a greater percentage of K562 cells had a subG1 DNA content when co-cultured with NK cells than when grown in the absence of NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, compare columns 3\u0026ndash;6 to column 2; Supplementary Fig. S6). The extent of the increase in the population of K562 cells with a subG1 DNA content was more pronounced as the abundance of NK cells increased relative to their target cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, compare columns 3\u0026ndash;6 to column 2; Supplementary Fig. S6). The length of the incubation period increased the population of K562 cells with a subG1 DNA content when the NK:K562 ratio was very high (4:1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, compare for example column 6 between panels; Supplementary Fig. S6). Taken together, our findings suggest that NK cells promote the generation of non-viable K562 cells with a subG1 DNA content in a cell number- and time-dependent manner.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA disruption coincides with immunocytotoxicity and cell death in NK cell-treated K562 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur earlier work demonstrated that RNA disruption coincides with cytotoxicity and cell death in tumour cells treated with chemotherapeutic drugs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We thus hypothesized that this relationship holds true in NK cell-treated K562 cells. We compared NK cell-mediated RNA disruption in K562 cells (quantified using the RDA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) with K562 cell immunocytotoxicity (measured as a loss of plasma membrane integrity using live/dead cell labelling) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and K562 cell death (measured as the generation of K562 cells with a subG1 DNA content using flow cytometry) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig. S6).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePre-treatment of K562 cells with doxorubicin enhances NK cell-mediated RNA disruption in K562 cells.\u003c/b\u003e An earlier study looking at the effect of chemotherapeutic drugs on tumour cells found that doxorubicin triggers RNA disruption in K562 cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Given that immunotherapy drugs like pembrolizumab have been shown to improve outcome from chemotherapy in early and late stage triple negative breast cancer\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and in metastatic non-small cell lung cancer\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and they are commonly prescribed along with chemotherapy, we next investigated whether we could improve NK cell-mediated RNA disruption by pre-treating K562 cells with doxorubicin prior to addition of NK cells. To this end, we pre-treated K562 cells with doxorubicin, then incubated the doxorubicin-treated K562 cells with NK cells in doxorubicin-free medium. We then removed NK cells from co-cultures using CD56\u0026thinsp;+\u0026thinsp;selection, isolated total RNA from remaining intact and lysed K562 cells and quantified RNA disruption using the RDA.\u003c/p\u003e \u003cp\u003eWe found that 1 and 10 \u0026micro;M doxorubicin, in the absence of subsequent treatment with NK cells, triggered RNA disruption and reduced RNA abundance in K562 cells, with greater effects achieved with higher drug doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, compare lanes/columns 9 and 13 to lane/column 5). Similarly, NK cells alone promoted RNA disruption and reduced RNA levels in K562 cells that were not exposed to doxorubicin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, compare lanes/columns 6\u0026ndash;8 to lane/column 5). Interestingly, when we co-cultured doxorubicin-treated K562 cells with NK cells, we detected a dramatic increase in RNA disruption in comparison to levels seen in K562 cells treated either with doxorubicin or NK cells alone. Of particular note, we found that levels of disruption seen in K562 cells treated with both doxorubicin and NK cells far exceeded the sum of disruption seen in cells treated with only the chemotherapeutic drug or only NK cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, compare for example lane/column 16 to lanes/columns 8 and 13). We also detected very low levels of RNA in K562 cells treated with both doxorubicin and NK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, columns 10\u0026ndash;12 and 14\u0026ndash;16). Taken together, our findings suggest that pre-treatment of K562 cells with doxorubicin augments the ability of NK cells to induce RNA disruption and reduce RNA abundance in K562 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNK cells and doxorubicin generate similar RNA disurption profiles within K562 cells.\u003c/b\u003e Since this study was the first to investigate immune cell-mediated RNA disruption, it was of interest to compare the abnormal RNA banding patterns of K562 cells treated with doxorubicin and those of K562 cells co-cultured with NK cells. We thus compared the RNA disruption profiles of both sample types by overlaying their electropherograms. Four pairs of samples with similar RDI values were used for comparison. We observed treatment-related reductions in the 28S and 18S rRNA peaks for both NK cell-treated and doxorubicin-treated K562 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), compared to untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In addition, the diffuse series of abnormal RNA peaks on electropherograms (particularly in the inter-region) appeared qualitatively similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Thus, regardless of contrasting mechanisms of cell death, doxorubicin and NK cells induce strikingly similar RNA disruption patterns in K562 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eFreshly isolated human NK cells are cytotoxic to K562 cells and induce both RNA disruption and cell death in K562 cells.\u003c/b\u003e We have confirmed in our study that NK cells are cytotoxic to K562 cells in culture, as measured by the ability of NK cells to compromise plasma membrane integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). NK cells also induce the death of K562 cells, as measured by strong increases in the number of K562 cells with a non-viable subG1 content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig. S6). We further show that the above phenotypic changes are accompanied by RNA disruption (higher RDI values) and reduced cellular RNA concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The magnitude of all four phenotypic changes is dependent upon the number of NK cells added to K562 cell preparations (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, the ratios of NK cells to K562 cells used to induce the above phenotypic changes were very similar to those employed in other studies. Kandarian \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e developed a flow cytometric cytotoxicity assay to assess human NK cell activity towards K562 cells. Their method was very similar to our loss of membrane integrity cytotoxicity assay. K562 cells were pre-labeled with a fluorescent dye to allow for discrimination from NK cells, and injured K562 cells with a compromised plasma membrane were identified using a cell-impermeable nucleic acid stain. A 4-h incubation period was also used in their study\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and the NK:K562 ratios ranged from 0.625 to 5, very similar to those used in our study. Using a similar flow cytometric approach, Kwoen \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e also demonstrated NK cell cytotoxicity towards K562 cells (using a 4-h incubation period and NK:K562 ratios identical to those used in this study). Other studies involving a \u003csup\u003e51\u003c/sup\u003eCr release assay\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e or image cytometry\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e employed identical incubation times but higher NK:K562 ratios. The above observations strongly suggest that the RNA disruption assay required similar parameters to other assays in order to demonstrate NK cell-mediated toxicity toward K562 cells.\u003c/p\u003e \u003cp\u003eThe mechanism by which NK cells are cytotoxic to K562 cells and induce their death is not entirely clear, but appears to involve the interaction between the NK62D and Ly49D receptors on the surface of NK cells with the Rae-I and H2D receptors on K562 cells\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. This results in the phosphorylation of motifs on the DNAX activation proteins Dap10 and Dap12 at the surface of NK cells, respectively, and subsequent activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway in NK cells. This in turn activates the transcription factors NF-κB and NFAT\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, which ultimately promotes the release of cytokines, chemokines and granzymes from NK cells and the release of perforins from their target cells\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The perforins then facilitate entry of granzymes into the target cell, which activates pro-apoptotic caspases\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In addition, the release of cytokines promotes apoptotic cell death through an interaction of Fas ligand on the surface of NK cells with Fas receptors on the surface of target cells\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere is some evidence that rRNA degradation may precede or accompany cell death in cells. For example, Houge and colleagues found that 8-(4-Chlorophenylthio)adenosine-3\u0026prime;,5\u0026prime;-cyclic monophosphorothioate strongly induces apoptosis in IPC-81 rat myeloid leukemia cells, and this was temporally correlated with the suppression of protein translation, internucleosomal DNA fragmentation, and the fragmentation of the 28S rRNA through cleavage at specific sites within its V3 and V13 variable regions\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. These investigators reported similar findings in NB4 myeloid leukemia cells treated with okadaic acid, rat thymocytes treated with prednisolone, and bovine endothelial cells treated with tumour necrosis factor and cycloheximide, except that the variable regions targeted during 28S rRNA fragmentation differed depending upon the apoptosis-inducing agent and the cell line used in experiments\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. rRNA degradation has also been shown to coincide with the induction of apoptosis in human lymphocytes undergoing starvation\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and in rodent thymocytes in response to many apoptosis-inducing cellular stressors\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Nadano \u003cem\u003eet al\u003c/em\u003e. demonstrated that activation of the Fas receptor with an anti-Fas antibody also induced both rRNA degradation and apoptosis in human U937 myeloid leukemia cells treated with tumour necrosis factor alpha\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. However, in one of these studies\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, rRNA degradation was found to occur in a caspase/Bcl-2-independent manner, suggesting that apoptotic pathways may not be required for rRNA degradation to occur.\u003c/p\u003e \u003cp\u003eIn our studies, though we observed RNA disruption reproducibly when K562 cells were treated with NK cells from different healthy volunteers, we found important variation in the degree of RNA disruption occuring in treated K562 cells. This variation may be due to several factors. Our donors were from both sexes and of varying age. NK cell activity has been found to vary by sex, with men having greater NK cytotoxicity than women\u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. In addition, NK cell cytotoxicity also varies with age, with older persons having lower cytoxocity than younger persons\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Other factors also appear to affect NK cell cytotoxicity, including obesity\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e and smoking\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Only non-smokers were invited to provide peripheral blood for this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePre-activation of NK cells with IL-2 augments NK cell-mediated RNA disruption of K562 cells.\u003c/b\u003e It has been shown that long-term incubation of NK cells with IL-2 (up to 6 days) enhances NK cell cytotoxicity towards target cell lines, although the same effect can be observed over shorter-term incubations of up to 24 h\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Moreover, similar to our study, Somanchi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e observed (using image cytometry) that primary human NK cells were cytotoxic to K562 cells after 4 h of incubation (although at an NK:K562 ratio of 10:1). The authors also observed that pre-stimulation of the NK cells with 50 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IL-2 resulted in complete lysis of K562 cells after 4 h at an NK:K562 ratio of only 2:1\u003csup\u003e54\u003c/sup\u003e. Zamai \u003cem\u003eet al.\u003c/em\u003e used both flow cytometry and a \u003csup\u003e51\u003c/sup\u003eCr-release assay to demonstrate significantly greater NK cell-mediated cytotoxicity against K562 cells when NK cells were pre-incubated overnight with 100 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IL-2 compared to unstimulated NK cells\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Clinically, the combination of IL-2 with ipilimumab, an inhibitor of the inhibitory T cell receptor CTLA-1, has been shown to increase survival in patients with metastatic melanoma compared to ipilimumab on its own\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Furthermore, Lotzova \u003cem\u003eet al\u003c/em\u003e. demonstrated that the NK cell impairment characteristic of leukemia patients can be reversed in culture with IL-2, and that fully cytotoxic NK cells can be maintained and expanded \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. What is the mechanism by which IL-2 improves NK cell cytotoxicity? Lehmann \u003cem\u003eet al.\u003c/em\u003e found that pre-activation of primary human NK cells with IL-2 resulted in increased K562 and ML-2 leukemia cell death due to improved binding of perforin to tumour cell membranes and subsequent lysis of tumour cells\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. This perforin-dependent mechanism for potentiation of NK cell cytotoxicity by IL-2 was also observed in another study using K562 target cells, where \u003cem\u003ein vitro\u003c/em\u003e pre-incubation of NK cells with IL-2 for 6 and 24 h corrected for a perforin deficiency that promoted reduced NK cell efficacy in people over the age of 70\u003csup\u003e76\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssociation of NK cell-mediated RNA disruption with NK cell-induced cell death in K562 cells.\u003c/b\u003e We found that NK cell-dependent RNA disruption in K562 cells was associated with cell death, as defined by an increase in the percentage of K562 cells with a non-viable subG1 DNA content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig. S6). These findings are consistent with our previously published works showing that chemotherapeutic drug-dependent RNA disruption is associated with the onset of cell death\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, unlike the clonogenic, trypan blue exclusion and cell counting kit-8 assays, which respond to low, sub-lethal concentrations of drugs that typically only induce cell cycle arrest, the RDA responds solely to drug concentrations that cause cell destruction (as defined by reduced cell numbers)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This may explain why high breast tumour RNA disruption during neoadjuvant chemotherapy is associated with complete tumour destruction (pCR) and improved disease-free survival after treatment\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The RDA is currently being assessed in a multi-institutional international clinical trial (BREVITY) for its ability to predict outcome from neoadjuvant chemotherapy in patients with breast cancer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clinicaltrials.gov/ct2/show/\u003c/span\u003e\u003cspan address=\"https://clinicaltrials.gov/ct2/show/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e NCT03524430).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDoxorubicin enhances NK cell-mediated RNA disruption and cell death in K562 cells.\u003c/b\u003e Previous investigations clearly show that treating tumour cells with low doses of chemotherapy agents can augment immune cell killing. For example, Borrelli \u003cem\u003eet al.\u003c/em\u003e observed that treating multiple myeloma cells with sub-lethal doses of doxorubicin and melphalan (an alkylating agent) led to senescence in these cells, causing increased expression of IL-15 and NK cell proliferation. Direct or exosome-mediated IL-15 trans-presentation to NK cells resulted in increased NK cell activation and proliferation, and thus, enhanced NK cell-tumour immune surveillance\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Soriani \u003cem\u003eet al\u003c/em\u003e. also observed that mouse melanoma cells treated with low doses of doxorubicin, melphalan and bortezomib exhibited a stress-induced senescent phenotype that stimulated the expression of NKG2D and DNAM-1 on their cell surface, which are well known NK cell-activating ligands\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. This was further supported by an additional study in a mouse melanoma model where low doses of melphalan promoted the establishment of a senescent tumour cell population with higher cell surface expression of NKG2D and DNAM-1, and greater recognition by NK cells \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. A similar phenomenon was observed with the chemotherapy agent 5-fluorouracil, which increased the expression of MHC I and NKG2D on Panc02 pancreatic cancer cells in mice. NK cells isolated from these mice exhibited enhanced cytotoxicity against Panc02 cells\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChemotherapy agents have also been shown to augment NK cell-mediated tumour cell death by promoting expression of death receptors on the surface of tumour cells. Wennerberg \u003cem\u003eet al.\u003c/em\u003e demonstrated that pre-treatment of various human tumour cell lines for 16 h with low doses of doxorubicin (184\u0026ndash;920 nM) resulted in a 2.5-fold increase in NK cell-mediated tumour cell lysis through increased apoptosis-inducing TRAIL receptor signaling. This was further confirmed in a xenogeneic tumour-bearing mouse model, where NK cell-dependent delays in tumour progression were further prolonged in mice pre-treated with doxorubicin compared to untreated control mice\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the close association between RNA disruption and cell death\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, it is therefore not surprising that pre-incubation of K562 cells with doxorubicin in our study augmented NK cell-mediated RNA disruption.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNK cells and doxorubicin induce similar RNA disruption patterns.\u003c/b\u003e Interestingly, NK cell-dependent changes to the RNA banding profile of K562 cells are qualitatively very similar to those resulting from doxorubicin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This suggests that despite the contrasting mechanisms by which doxorubicin and NK cells promote the death of tumour cells, they both ultimately induce RNA disruption. Taken together with our previously published findings that different chemotherapy drugs and cellular stressors induce RNA disruption \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and/or \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e, our work here suggests that RNA disruption may be a hallmark feature of dying tumour cells. However, the time required to evoke substantial RNA disruption appears to be much shorter for NK cells (4 h in this study) than for doxorubicin (48\u0026ndash;72 h, depending upon the drug dose)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. How do various chemotherapy drugs, cellular stressors and NK cells all trigger RNA disruption? One possibility is the ability of chemotherapy agents\u003csup\u003e\u003cspan additionalcitationids=\"CR84\" citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e, cellular stressors\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e and granzymes released by NK cells\u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e to generate reactive oxygen species (ROS) in tumour cells. These ROS can strongly reduce ribosome catalytic activity\u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e and promote a variety of mutations in rRNAs, including guanine base oxidation, strand scission and rRNA-protein cross-links (recently reviewed by Shcherbik and Pestov\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e, and Tanaka and Chock\u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e). ROS generation can then promote RNA disruption by causing mutations in rRNAs, which render them non-functional and activate the 28S/18S non-functional RNA decay\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e and ribophagy\u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e pathways. In addition, the generation of ROS can promote cell death by oxidizing permeability transition pore channels within mitochondria, resulting in the release of a variety of pro-apoptotic factors\u003csup\u003e\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e. Protein oxidation within the endoplasmic reticulum can induce the unfolded protein response\u003csup\u003e\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e, which if left unchecked, activates pro-apoptotic pathways\u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e. Whereas transient activation of the non-functional rRNA decay and ribophagy pathways may permit cells to survive exposure to cellular stressors and chemotherapy agents by reducing the energetically costly process of protein translation, prolonged activation of these pathways appears to promote cell death through organelle destruction and the activation of cell death pathways.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePossible use of the RDA to monitor response to immune checkpoint inhibitory drugs.\u003c/b\u003e Recent evidence suggests that the addition of immune checkpoint inhibitors to cytotoxic chemotherapy regimens strongly improves treatment outcome for patients with a variety of cancers. For example, the combination of the PD1 inhibitor pembrolizumab with chemotherapeutic drugs carboplatin and paclitaxel has now been adopted as first-line therapy in patients with metastatic squamous non-small cell lung carcinoma\u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Moreover, the combination of pembrolizumab with doxorubicin and cyclophosphamide has been shown to significantly improve pCR rates in patients with high-risk breast cancer relative to standard neoadjuvant chemotherapy alone\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven that NK cells have been shown in this study to induce RNA disruption in K562 tumour cells (which can be augmented by pre-treatment of the K562 cells with doxorubicin), and that this RNA disruption is reproducibly associated with the onset of tumour cell death, we are now assessing whether the RDA can also be used to predict patient response and outcome from treatments involving immune checkpoint inhibitory drugs (alone or in combination with cytotoxic chemotherapy drugs). Unlike this study, where the amount of RNA isolated from K562 cells at very high doxorubicin doses or high NK:K562 co-culture ratios precluded our ability to compute RDI values, we have observed in the BREVITY clinical trial that tumour RNA yields from on-treatment biopsies of breast cancer patients appear to be sufficiently high in the vast majority of cases to obtain RDI values for treatment outcome prediction (manuscript in preparation).\u003c/p\u003e \u003cp\u003eIn summary, we have observed in this study that K562 chronic myeloid leukemia cells treated with NK cells exhibit high RNA disruption (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a loss of plasma membrane integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and increased subG1 DNA content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig. S6), suggesting a strong positive association between RNA disruption, immunocytotoxicity and cell death in NK cell-treated K562 cells. Pre-incubation of K562 cells with doxorubicin augmented NK cell-induced RNA disruption (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b) and NK cell-induced reductions in RNA concentration in K562 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). One of the previously recognized advantages of the RDI as a predictive biomarker when administering chemotherapy drugs is that it can predict complete tumour cell destruction post-treatment (pCR) when measured after only one cycle of chemotherapy\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Our findings in this study raise the prospect that the RDA may be similarly useful in predicting early in treatment patient outcome from immunotherapies, possibly with or without co-administration of chemotherapy drugs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eCell culture.\u003c/b\u003e The non-adherent K562 human chronic myeloid leukemia cell line was selected for this investigation since it is sensitive to NK cell-mediated killing due to very low expression of MHC class I\u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e. It was purchased from the American Type Culture Collection. K562 cells were maintained in IMDM medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Gibco). K562 cells were cultured in 75-cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e vented flasks for suspension cells (Sarstedt) in a humidified, water-jacketed incubator set to 37\u0026deg;C and 5% CO2. Cells were allowed to proliferate to a maximum density of 1\u0026nbsp;million cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e prior to subculture (every 3\u0026ndash;4 days). K562 cultures were confirmed to be free of \u003cem\u003eMycoplasma\u003c/em\u003e infection by performing polymerase chain reaction experiments using a \u003cem\u003eMycoplasma\u003c/em\u003e detection kit from Applied Biological Materials.\u003c/p\u003e \u003cp\u003e\u003cb\u003eRecruitment of blood donors for isolation of NK cells.\u003c/b\u003e Blood donors were recruited following the study protocol approved by Laurentian University\u0026rsquo;s Research Ethics Board (no. 6016116) and the Health Sciences North Research Ethics committee. The studies involving human volunteers were performed in accordance with the Canadian TriCouncil policies on ethical conduct for research involving humans (TCPS2 2022). Each donor provided written informed consent prior to participation in this study. Healthy volunteers were recruited from Sudbury, ON, Canada through advertisements to staff and students associated with Laurentian University, Health Sciences North and the Northern Ontario School of Medicine. Inclusion criteria for donors were: persons between 20\u0026ndash;59 years old, weight over 54 kg, non-smoker, no prior history of anemia, no chronic inflammatory diseases, and no regular use of anti-inflammatory drugs. A registered phlebotomist collected 50\u0026ndash;60 mL of blood from each donor once a week for up to 5 weeks. Venous whole blood from each volunteer was collected in seven 10-mL Vacutainer heparinized blood collection tubes (BD), and tubes were inverted several times to ensure proper mixing. Eleven donors provided whole blood for this study. Samples from 2 donors were used for optimization of experimental procedures, after which blood cells from 9 donors were used for the research study. Multiple replicate experiments involving a minimum of 3 independent donors were performed to obtain the data depicted in this investigation. The protocols used for both blood collection and for the performance of all described experiments were approved by Health Sciences North\u0026rsquo;s Biohazard Safety Committee.\u003c/p\u003e \u003cp\u003e\u003cb\u003eIsolation of human peripheral blood mononuclear cells (PBMCs)\u003c/b\u003e. PBMCs were isolated from human whole blood using SepMate-50 tubes (Stemcell Technologies) following the manufacturer\u0026rsquo;s guidelines. The density gradient medium used was Ficoll-Paque PLUS density gradient media (GE Healthcare Life Sciences), and the wash and resuspension buffers consisted of EasySep Buffer (Stemcell Technologies). Platelets were removed from the recovered PBMC preparation as follows. PBMCs were centrifuged at 200 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min followed by a slow deceleration. After removal of the supernatant, the PBMC pellet was resuspended in 1 mL of EasySep Buffer. Cells were then counted using a haemocytometer.\u003c/p\u003e \u003cp\u003e\u003cb\u003eEnrichment of NK cells from the PBMC population\u003c/b\u003e. Typically, NK cells account for only 5\u0026ndash;20% of cells within the PBMC population\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. NK cells were thus enriched within the cell mixture using the EasySep\u0026trade; Direct Human NK Cell Isolation Kit (Stemcell Technologies) with the EasySep Magnet (Stemcell Technologies) following the manufacturer\u0026rsquo;s guidelines. The recommended medium (EasySep Buffer) was used. The recovered enriched NK cells were pelleted by centrifugation at 400 \u0026times; \u003cem\u003eg\u003c/em\u003e for 3 min and resuspended in 1 mL of IMDM medium supplemented with 10% FBS. Cells were then counted using a haemocytometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of purity of NK cell preparations by flow cytometry.\u003c/b\u003e Flow cytometry was performed to confirm that the purity of NK cells after enrichment from the PBMC population was \u0026gt;\u0026thinsp;80%. Because NK cells have the surface cell differentiation markers CD45 and CD56, but lack the surface cell differentiation marker CD3\u003csup\u003e99\u003c/sup\u003e, we used the following fluorochrome-conjugated antibody clones, all obtained from BD Biosciences, to calculate the percentage of cells with a CD56\u0026thinsp;+\u0026thinsp;CD3- CD45\u0026thinsp;+\u0026thinsp;expression profile: (i) PE-CF594 mouse anti-human CD45 (clone HI30), (ii) PE-Cy7 mouse anti-human CD56 (NCAM-1) (clone B159), and (iii) BB515 mouse anti-human CD3 (clone UCHT1). eBioscience Fixable Viability Dye (FVD) eFluor 780 (Invitrogen) was also included to irreversibly label dead cells. No staining, FVD staining, single-antibody staining and fluorescence-minus-one controls (where one antibody is removed at a time to reveal the amount of fluorescent spillover of other fluors in the panel into the left-out parameter) were used to determine gates and compensation for flow cytometric characterization of both PBMCs and enriched NK cells.\u003c/p\u003e \u003cp\u003ePBMCs and enriched NK cells were collected by centrifugation at 400\u0026ndash;500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min, and resuspended in 1 \u0026times; phosphate-buffered saline solution (PBS) without Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e (Cytiva, Hyclone) supplemented with (immunofluorescence staining) or without (FVD staining) 1% bovine serum albumin (BSA) to a final concentration of 10\u0026nbsp;million cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (immunofluorescence staining) or 1\u0026nbsp;million cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (FVD staining). Then, 1 \u0026micro;L of FVD or 50 \u0026micro;L of fluorochrome-conjugated antibody were added per mL of cell suspension. Mixtures were incubated in the dark for 15\u0026ndash;30 min at 4\u0026deg;C. Cells were then collected by centrifugation and washed with PBS containing 1% BSA. Cells were again pelleted and resuspended to a final concentration of 1\u0026nbsp;million cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in cold (4\u0026deg;C) PBS containing 1% BSA. Then, 200 \u0026micro;L of each sample were transferred to a 96-well tissue culture-treated microplate (Corning) and analyzed using the CytoFLEX LX flow cytometer with the CytExpert software (Beckman Coulter). A total of 10,000 events were measured per sample. NK cells were successfully enriched to at least 90% purity (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eActivation of NK cells with IL-2\u003c/b\u003e. NK cells freshly isolated from PBMCs were first resuspended in RPMI-1640 medium (HyClone) supplemented with 10% FBS. Immediately, NK cells were plated in 24-well tissue culture-treated flat-bottom plates (Sarstedt) at a density of 1\u0026nbsp;million cells per well in 1 mL of growth medium supplemented with or without 100 or 1,000 IU mL\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e recombinant human IL-2 (Peprotech). IL-2 concentrations were selected based on the most common doses used in prior experiments with NK cells\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e,\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/sup\u003e. Cultures were incubated at 37\u0026deg;C overnight (approximately 16 h). Cultures were then centrifuged at 400 \u0026times; \u003cem\u003eg\u003c/em\u003e for 3 min, and the cell pellets were resuspended in IMDM medium supplemented with 10% FBS to a final density of 4\u0026nbsp;million cells mL\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Cells were then used to treat K562 cells as described below.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePre-treatment of K562 cells with doxorubicin\u003c/b\u003e. Doxorubicin was kindly provided by the pharmacy at Health Sciences North (Sudbury, ON, Canada). K562 cells were plated in 6-well tissue culture-treated plates (Sarstedt) at a density of 250,000 cells per well in 3 mL of growth medium supplemented with 0, 1 or 10 \u0026micro;M doxorubicin. Suspension cultures were then incubated as described above for 48 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncubation of NK cells with K562 tumour cells.\u003c/b\u003e K562 suspension cultures, pre-treated with or without doxorubicin, were centrifuged at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min. The culture supernatant was discarded, and pelleted cells were resuspended in fresh drug-free IMDM medium supplemented with 10% FBS. One hundred microliters of K562 cell suspension (containing 100,000 cells) were mixed with 100 \u0026micro;L of unstimulated or IL-2-activated NK cell suspension (containing 50,000 to 400,000 cells to obtain NK:K562 ratios of 0.5:1, 1:1, 2:1 or 4:1) in a 96-well flat-bottom tissue culture-treated plate (Corning). \u0026ldquo;K562 cells only\u0026rdquo; and \u0026ldquo;NK cells only\u0026rdquo; controls were prepared in parallel. \u0026ldquo;K562 cells only\u0026rdquo; controls comprised 200 \u0026micro;L of K562 cell suspension (containing 100,000 cells) devoid of NK cells, whereas \u0026ldquo;NK cells only\u0026rdquo; controls consisted of 200 \u0026micro;L of NK cell suspension (containing 50,000 to 400,000 cells) lacking K562 cells. Cell mixtures were incubated at 37\u0026deg;C for 0 or 4 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRemoval of NK cells from K562 cells after incubation.\u003c/b\u003e When appropriate, the NK cells were removed from the NK/K562 co-cultures by a CD56\u0026thinsp;+\u0026thinsp;selection method using the EasySep Human CD56 Positive Selection Kit II (Stemcell Technologies) with the EasySep Magnet (Stemcell Technologies). Subsets of the \u0026ldquo;K562 cells only\u0026rdquo; and \u0026ldquo;NK cells only\u0026rdquo; controls were also submitted to CD56\u0026thinsp;+\u0026thinsp;selection using this method. Briefly, cultures were centrifuged at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min. The supernatants were removed and discarded, and the cell pellets were re-suspended in 100 \u0026micro;L of EasySep Buffer. Removal of the NK cells proceeded following the manufacturer\u0026rsquo;s instructions, with the following exceptions. The final supernatants (containing intact and/or lysed CD56-negative K562 cells) were recovered; the NK cells remaining in the tube (due to their binding to CD56-positive magnetic beads that adhered to an external magnet) were discarded. The recommended medium (EasySep Buffer) was used. The supernatant was then centrifuged at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min, and the pelleted cells were used for either DNA content analysis by flow cytometry or RNA integrity assessement, as described below.\u003c/p\u003e \u003cp\u003e\u003cb\u003eImmunocytotoxicity assays.\u003c/b\u003e The ability of NK cells to induce K562 cell cytotoxicity was assessed by monitoring the loss of plasma membrane integrity in K562 cells using the Incucyte S3 Live-Cell Analysis System (Sartorius) with the Incucyte Cytolight Rapid Red Dye (Sartorius) and the Incucyte Cytotox Green Dye (Sartorius) following a protocol adapted from the manufacturer\u0026rsquo;s guidelines. Briefly, K562 cultures were centrifuged at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min, and the resulting cell pellets were re-suspended and washed in 10 mL of PBS. Cells were collected by centrifugation as above and re-suspended in sufficient PBS to achieve a cell density of 1\u0026nbsp;million cells mL\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. To label live K562 cells, the cell-permeable, red-fluorescing Incucyte Cytolight Rapid Red Dye was added to the cell suspensions at a final concentration of 3 \u0026micro;M. The mixtures were incubated at 37\u0026ordm;C for 20 min with periodic mixing. Excess dye was then removed by adding 6-fold excess volume of IMDM containing 10% FBS and harvesting the cells by centrifugation as above. Pelleted Rapid Red Dye-labelled cells were re-suspended in IMDM supplemented with 10% FBS. Then, 50 \u0026micro;L of K562 cell suspension (containing 50,000 cells) and 50 \u0026micro;L of NK cell suspension (containing 25,000-200,000 cells) were plated in 96-well flat-bottom plates (Corning). For \u0026ldquo;K562 cells only\u0026rdquo; and \u0026ldquo;NK cells only\u0026rdquo; controls, 100 \u0026micro;L of the appropriate cell suspension (containing an equivalent number of cells) were plated. After plating cells, Incucyte Cytotox Green Dye, a cell-impermeable, green-fluorescing dye used to label cells with compromised plasma membranes, was added directly to wells to yield a final dye concentration of 250 nM. Cells were permitted to settle to the bottom of the plate for 20\u0026ndash;30 min prior to imaging. Plates were then placed in the Incucyte S3 Live-Cell Analysis System at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for imaging at 10 \u0026times; magnification and counting using Incucyte\u0026rsquo;s 2018C software every 15 min for 4 h. Labelled NK cells were excluded from K562 cell counts using the instrument\u0026rsquo;s size filter feature, as NK cells are considerably smaller than K562 cells (Supplementary Figs. S2 and S4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA content analyses by flow cytometry.\u003c/b\u003e The ability of NK cells to induce K562 cell death was assessed by quantifying K562 cells with a subG1 DNA content using a flow cytometry procedure adapted from Butler \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Briefly, 50,000 K562 cells with or without (\u0026ldquo;K562 cells only\u0026rdquo; controls) 25,000-200,000 NK cells were cultured for 0, 4, 24 and 48 h in 800 \u0026micro;L of IMDM supplemented with 10% FBS in 24-well flat-bottom plates (Sarstedt). Cultures were centrifuged at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min. Pelleted cells were then resuspended and washed twice with 2 mL of PBS, and collected by centrifugation at 233 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min. Next, cell pellets were re-suspended in 0.5 mL of PBS and placed on ice. Cells were immediately fixed by mixing 1.5 mL of cold (-20\u0026deg;C) anhydrous ethanol with the cell suspensions. Fixed cells were stored at -20\u0026deg;C until further use. Fixed cell suspensions were centrifuged at 530 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min. Cell pellets were resuspended and washed with 2 mL PBS, and again harvested by centrifugation. Cells were stained by re-suspending the pellets with 500 \u0026micro;L of propidium iodide staining solution (100 \u0026micro;g mL\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e propidium iodide, 100 \u0026micro;g mL\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e RNase A, 0.3% NP-40, and 0.3% sodium citrate) and incubating the suspensions at 37\u0026deg;C for 30 min. Stained cells were then analyzed using a Cytomics FC500 flow cytometer (Beckman Coulter) with the 675-nm bandpass filter (488-nm excitation wavelength). Samples were measured for 20,000 events or 300 sec, whichever occurred first. Data analyses were performed using the CXP Analysis software (Beckman Coulter) without the use of gating.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA disruption assay\u003c/b\u003e. K562 cells were collected from cultures by centrifugation at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min. Total RNA was then isolated from pelleted cells using the RNeasy Mini kit (Qiagen) following the manufacturer\u0026rsquo;s protocol titled \u003cem\u003ePurification of Total RNA from Animal Cells using Spin Technology.\u003c/em\u003e Cells were homogenized using a needle, as described by the manufacturer. RNA concentrations were measured using the NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific). Total RNA was resolved by capillary gel electrophoresis using the 2100 Bioanalyzer (Agilent Technologies) with the RNA 6000 Nano kit (Agilent Technologies) per the manufacturer\u0026rsquo;s instructions. RNA electropherogram data files were transmitted electronically to Rna Diagnostics for RDI calculation. The RNA electropherograms depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea are composites of individual experiments conducted using 12 well RNA Nanochips\u003csup\u003e\u0026trade;\u003c/sup\u003e and are representative of three independent experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGraphics.\u003c/b\u003e Plots were prepared using Prism. Electropherograms were generated using the 2100 Expert software version B.02.09.SI725 (SR1) (Agilent Technologies). Flow cytometry plots for purity assessment of NK cell preparations and DNA content analyses experiments were produced using the CytExpert software (Beckman Coulter) and the CXP Analysis software (Beckman Coulter), respectively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors would like to thank Drs. Carita Lanner and Tom Kovala (NOSM University and Laurentian University, Sudbury, ON, Canada), as well as Dr. Hoang-Thanh Le (Health Sciences North Research Institute, Sudbury, ON, Canada) for their input into the design of studies, the interpretation of experiments, and/or critical feedback on the manuscript. \u0026nbsp;They would also also like to thank Dr. Graham Pawelec (University of T\u0026uuml;bingen,\u0026nbsp;T\u0026uuml;bingen, Germany) for providing guidance on the design of immunocytotoxicity experiments and Ren\u0026eacute;e St. Onge for her throughout editorial review of the manuscript prior to submission. \u0026nbsp; \u0026nbsp;This work was supported by a MITACS Accelerate graduate student internship award (IT2731) to I.P. and a grant from the Northern Cancer Foundation (Sudbury, ON, Canada) to A.P., along with in-kind support from Rna Diagnostics (Sudbury and Toronto, ON, Canada).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA.M.P. and A.K. raised funds and played a role in hypothesis generation and the design of experiments. A.K. served as the academic supervisor for this MITACS-funded internship project. I.P. and A.M.P. wrote the manuscript, and all authors participated in its review. I.P. conducted all of the experiments and prepared the manuscript figures. B.G. advised I.P. on experimental protocols and use of the live-cell imaging system and flow cytometer. L.B.P. computed RDI values for all samples. \u0026nbsp;All authors reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMembers of the team disclose the following significant conflicts of interest. A.M.P. holds a large number of shares in Rna Diagnostics, Inc., a company that is commercializing the RDA for clinical use. He also serves as the company\u0026rsquo;s Chief Scientific Officer. L.B.P. is Vice President of Research and Development for Rna Diagnostics. A large portion of B.G.\u0026rsquo;s salary and benefits was supported by Rna Diagnostics during this investigation. A.M.P. and B.G. have and will receive royalties from the commercialization of the RDA. I.P and \u003cstrong\u003eA.K. have no competing interests to disclose.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eSupplementary figures, and electropherogram files used to create manuscript figures can be found in the supplementary information pdf file ESM_1. Excel spreadsheets containing quantitative data from all electropherograms (including RDI and RNA concentration values), live (red)/dead (green) cell counting data, and counts of cells at various stages of the cell cycle can all be found in the supplementary dataset Excel (xlsx) file ESM_2.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang, J., \u003cem\u003eet al.\u003c/em\u003e Efficacy and safety of neoadjuvant therapy for HER2-positive early breast cancer: a network meta-analysis. Ther Adv Med Oncol 13, 17588359211006948 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbain, K.S., \u003cem\u003eet al.\u003c/em\u003e Adjuvant chemotherapy and timing of tamoxifen in postmenopausal patients with endocrine-responsive, node-positive breast cancer: a phase 3, open-label, randomised controlled trial. 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Theranostics 8, 3856\u0026ndash;3869 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RNA disruption, ribosomal RNA degradation, chemotherapy, immunotherapy, natural killer cells, plasma membrane integrity, cell death, DNA content","lastPublishedDoi":"10.21203/rs.3.rs-2944450/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2944450/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtensive degradation of tumour 28S and 18S ribosomal RNAs, coupled with the accumulation of ribosomal RNA degradation products, is associated with pathologic complete response and improved disease-free-survival in breast cancer patients. Various chemotherapy agents and cellular stressors are known to trigger this process, termed \u0026lsquo;RNA disruption\u0026rsquo;, in tumour cells. However, it\u0026rsquo;s unclear whether immunotherapies, with or without chemotherapy administration, also trigger RNA disruption. To address this question, we assessed the ability of natural killer (NK) cells to induce RNA disruption and cell death in K562 chronic myeloid leukemia cells \u003cem\u003ein vitro\u003c/em\u003e. We found that NK cells strongly stimulated RNA disruption, cytotoxicity (loss of plasma membrane integrity) and cell death (generation of cells with a subG1 DNA content) in K562 cells. Pre-activation of NK cells with interleukin-2 or pre-treatment of K562 cells with the chemotherapy drug doxorubicin augmented RNA disruption in K562 cells. RNA degradation patterns looked very similar between NK cell-treated and doxorubicin-treated K562 cells. Our observations suggest that RNA disruption is strongly associated with cell death irrespective of the death-inducing stimulus and raise the prospect that tumour RNA disruption may be a useful biomarker for quantifying cancer patients\u0026rsquo; response to immunotherapies, with or without co-administration of chemotherapy drugs.\u003c/p\u003e","manuscriptTitle":"Association of extensive RNA disruption with natural killer cell-mediated death of K562 chronic myelogenous leukemia cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-05 14:37:38","doi":"10.21203/rs.3.rs-2944450/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f68c1468-44e2-418d-bd7f-aef019959d36","owner":[],"postedDate":"June 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":22014741,"name":"Biological sciences/Biological techniques"},{"id":22014742,"name":"Biological sciences/Cancer"},{"id":22014743,"name":"Biological sciences/Immunology"},{"id":22014744,"name":"Biological sciences/Molecular biology"},{"id":22014745,"name":"Health sciences/Biomarkers"},{"id":22014746,"name":"Health sciences/Molecular medicine"},{"id":22014747,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2023-06-27T02:59:27+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-05 14:37:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2944450","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2944450","identity":"rs-2944450","version":["v1"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}