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Bioimprint aided cell recognition and depletion of human leukemic HL60 cells from peripheral blood

Das, Anupam A.K.; Medlock, Jevan; Liang, He; Nees, Dieter; Allsup, David J.; Madden, Leigh A.; Paunov, Vesselin N.

Authors

Anupam A.K. Das

Jevan Medlock

He Liang

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Dr David Allsup D.J.Allsup@hull.ac.uk
Senior Lecturer in Haematology and Honorary Consultant

Leigh A. Madden

Abstract

We report a large scale preparation of bioimprints of layers of cultured human leukemic HL60 cells which can perform cell shape and size recognition from a mixture with peripheral blood mononuclear cells (PBMCs). We demonstrate that the bioimprint-cell attraction combined with surface modification and flow rate control allows depletion of the HL60 cells from peripheral blood which can be used for development of alternative therapies of acute myeloid leukaemia (AML).

AML is a clonal malignant proliferation of transformed, bone-marrow derived myeloid precursors. The disease is characterised by the rapid proliferation of the neoplastic cells (myeloblasts) resulting in failure of normal haematopoiesis with consequential bone marrow failure rapidly resulting in death if untreated.1–3 In the UK, overall survival is 16% 5 years from diagnosis. The prognosis is significantly worse in the elderly which is especially relevant as the majority of patients present over the age of 60 years.1,4–7 Therapy relies on 2–3 cycles of myeloablative chemotherapy followed by allogeneic stem cell transplants for a relatively small number of fit patients with poor prognostic features.8,9 This is accompanied by significant discomfort, and long therapy for AML is also associated with prolonged inpatient stays, considerable morbidity related to anaemia, sepsis and bleeding with an attributable mortality of 5–10%. The majority of patients relapse following induction of chemotherapy for AML and subsequent therapy is associated with a low probability of cure. Outcomes for AML patients have improved marginally over the past few decades, largely due to improvements in supportive care rather than dramatic improvements in the chemotherapeutic regimen's efficacy.10

Bioimprinting is a promising area of materials chemistry aimed at mimicking and exploiting the lock-and-key interactions seen ubiquitously in nature.11–14 Cell recognition systems are relatively cheap and simple to produce with few stipulations on storage and shelf life when compared with biological interventions. The scope for possible targets is also much greater, being able to target polysaccharides, enzymes, aptamers, DNA sequences, antibodies and whole cells.12,15,16,21–24 Bioimprints of whole cells were first reported by Dickert et al.17 who imprinted yeast into a sol–gel matrix. When incubated with several strains of yeast, the substrates showed a high affinity to the template yeast strain. This effect was attributed to the large contact surface areas between the cells and the imprinted cavities. Other cell bioimprinting studies have progressed to cover a range of micro-organisms and human cells. Hayden et al.18 functionalised polyurethane with erythrocyte imprints, capable of discriminating between ABO blood groups. Though all cell targets possessed the same geometrical shape and size, imprints were able to discriminate on account of varied surface antigen expression. Subsequent studies were further able to discriminate cells with identical antibodies in different quantities to separate blood groups A1 and A2.19 Recent cell bioimprint studies largely focus on biosensor applications20,26 and are hindered by the small overall size of imprinted areas that can be produced which limits their applications for large scale extraction of targeted cells from cell mixtures. This research area is undergoing a rapid expansion towards using molecularly imprinted polymers as receptor mimics for selective cell recognition and sensing, and a recent review of size and shape targeting of cancer found no evidence so far of the use of cancer cell bioimprints in a therapeutic setting.11

Here we utilised for the first time AML cell bioimprints on a large scale as a vehicle to selectively target myeloblasts due to the inherent size and morphological discrepancies compared to normal peripheral blood mononuclear cells (PBMCs) (see Fig. S1, ESI†). We explore AML cells bioimprinting to develop a new method for depletion of myeloblasts from peripheral blood cells by introducing selectivity via bespoke cell size and shape discrimination aided by myeloblast-bioimprint interactions. Our idea is based on incorporating AML cells-imprinted substrates into a flow-through type of device which offers an alternative method for removal of the leukemic burden directly from patient blood. Successful leukophoresis can potentially be used more frequently in the extraction of myeloblasts from peripheral blood which is critical in stabilizing AML patients with leukostasis associated with hyperleuocytosis. By reducing the number of circulating tumour cells, the likelihood of early relapse is also diminished.25

HL60 is an immortalized human cell line derived from peripheral blood lymphocytes of a patient suffering from acute promyleocytic leukaemia. HL60 was used as a very good proxy for primary (patient derived) myeloblast cells throughout our study due to their availability and ease of culture. Here we show how the desired HL60 cell bioimprints were produced from HL60 cell layers. We also discuss the integration of the produced myeloblast imprint in a PDMS-based flow-through cell, in which its selectivity towards HL60 cells over PBMCs is investigated (Fig. 1). We fabricated bioimprints by impressing a layer of cultured HL60 cells with a curable polymer, which captures information on the cell shape, size and morphology. These were further casted with another polymer to create a “positive imprint” whose surface matches the original cell layer. Using roll-to-roll printing from the positive replica we produced a very large area of HL60 cell imprints. We engineered the surface of the bioimprint to have a weak attraction with the cells, which is strongly amplified when there is a shape and size match between the individual cells and the imprinted surface. Due to inherent size and morphology differences between myeloblasts and normal blood cells, this resulted in much higher retention of the former on the bioimprint. This allows their selective trapping from peripheral blood based on cell shape and size recognition, much cheaper than using surface functionalisation with a combination of specific antibodies for myeloblasts. We tested the bioimprints selectivity in a device for depleting cultured HL60 cells from healthy white blood cells. This cell recognition technology can potentially deplete myeloblasts from the blood of AML patients and provide an alternative route for inducing minimal residual disease, which is associated with reduced relapses and improved patient outcomes.

Journal Article Type Article
Journal Journal of Materials Chemistry B
Print ISSN 2050-750X
Electronic ISSN 2050-7518
Publisher Royal Society of Chemistry
Peer Reviewed Peer Reviewed
DOI https://doi.org/10.1039/c9tb00679f
Keywords General Materials Science; General Chemistry; Biomedical Engineering; General Medicine
Publisher URL https://pubs.rsc.org/en/content/articlelanding/2019/TB/C9TB00679F#!divAbstract
Additional Information This is the accepted manuscript of an article published in Journal of Materials Chemistry B , 2019. The version of record is available at the DOI link in this record.