Adoptive cell therapies require the recovery and expansion of highly potent tumour-infiltrating lymphocytes (TILs). However, TILs in tumours are rare and difficult to isolate efficiently, which hinders the optimization of therapeutic potency and dose. Here we show that a configurable microfluidic device can efficiently recover potent TILs from solid tumours by leveraging specific expression levels of target cell-surface markers. The device, which is sandwiched by permanent magnets, balances magnetic forces and fluidic drag forces to sort cells labelled with magnetic nanoparticles conjugated with antibodies for the target markers. Compared with conventional cell sorting, immunomagnetic cell sorting recovered up to 30-fold higher numbers of TILs, and the higher levels and diversity of the recovered TILs accelerated TIL expansion and enhanced their therapeutic potency. Immunomagnetic cell sorting also allowed us to identify and isolate potent TIL subpopulations, in particular TILs with moderate levels of CD39 (a marker of T-cell reactivity to tumours and T-cell exhaustion), which we found are tumour-specific, self-renewable and essential for the long-term success of adoptive cell therapies.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. The unprocessed TCR sequencing files are too large to be publicly shared, but they are available from the corresponding author on reasonable request. Source data are provided with this paper.
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We thank J. Charron and N. Simard at the Temerty Faculty of Medicine, University of Toronto for help in FACS sorting; T. Chen at the Sick Children Hospital, Toronto for help in tumour dissociation; M. Peralta at the University Health Network (UHN) for help in immunohistochemistry; W. Xiao at UHN for help in intravascular injection; J. Henderson at the Faculty of Pharmacy, University of Toronto; J. Cathcart and J. Jonkman at UHN for help in image quantitation; and anonymous technician(s) at Miltenyi Biotec for information regarding the MACSQuant Tyto system. This research was supported in part by the Canadian Institutes of Health Research (grant no. FDN-148415) and the Collaborative Health Research Projects program (CIHR/NSERC partnered). This research is part of the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund. Z.W. was supported by an Alexander Graham Bell Canada Graduate Scholarship and a Centre for Pharmaceutical Oncology Graduate Student Scholarship.
S.O.K., Z.W. and S.A. have a filed patent application (number 63/183,350) using parts of the data reported in this article. S.O.K. has a patent titled ‘Device for capture of particles in a flow’ (US10073079) licensed to Cellular Analytics. S.O.K. received research funds from Amgen through a sponsored research agreement. J.M. is a shareholder of Century Therapeutics and Aelian Biotechnology.
Peer review information Nature Biomedical Engineering thanks Paul Robbins and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Principle, fabrication and assembly of modular devices for configurable microfluidic cell sorting.
a, Simulated flow velocity distribution within the devices with different heights, unit of colour bar (m·s-1). Capture pockets were form nearly the middle of ‘X’-shaped structures spatially. b, Quantitation of the simulated flow velocity in different cross-sections. In a cross-section that travel through the middle of the ‘X’-shaped structures, the flow velocity in the capture pocket was extremely low (<1% of the maximal) to favour the cell capture. In a cross-section that did travel through the X, the flow velocity remains high and no capture pocket was formed. c, Representative SEM images of various printed ‘X’-shaped structures with heights up to 800 µm. All designed features were printed properly without any major defects. d, Representative pictures showing the fabricated modular devices. A red food dye was used to visualize the change of heights. All ‘X’-shaped structures were properly bonded to the cover glass, as shown in the zoom-in picture. e, Representative pictures showing the key components of configurable microfluidic sorting, including the fabricated modular chips, magnetic scaffolds, and a finished quantitative sorting setup.
Extended Data Fig. 2 Cytotoxicity and cytokine profile of different TILs through in vitro co-culture killing assay.
a, Quantitation of cytotoxic killing of TILs against B16F10OVA cells in vitro. b, Cytokine profile of the supernatant collected from in vitro killing assay. Pure B16F10-OVA is used as an internal control.
Extended Data Fig. 3 MATIC TILs are more potent in treating rapidly developing melanoma in vivo.
a, Workflow of the study comparing the therapeutic efficacy of TILs isolated by different methods, at the optimal dosage ~5 ×105 at its earliest (D5 for MATIC, D10 for MACS, D15 for FACS, for FACS, lower number (5 ×104) of TILs were injected as it fails to reach desired concentration before mouse of mice developed large tumours). b, Representative tumour size of each group on D18. c, Quantitation of tumour size and survival curve treated by the TILs isolated by MATIC, MACS and FACS (n = 5, **P < 0.01). Log-rank test was used to determine the statistical significance. d, Representative images of infiltrated T cells in solid tumours (Blue: nuclei, Red: CD8α, Brown: melanin). e, Quantitation of the number of CD8+ TILs in the tumours treated by MATIC, MACS and FACS TILs (n = 3, 2 - 3 slices per tumour). f, Tumour growth curves for each mice presented in Supplementary Fig. 14C. PFS: Progression-free survival.
Extended Data Fig. 4 Flow cytometric analysis of TILs from different CD39 populations in a MC-38 mouse model.
a, MC-38 model has about 1% CD8 + TILs within the tumours according to CD8/CD45 gating. b, Representative cytometric profile and quantitation of exhaustion markers (PD-1, TIM3, TIGIT). c, Representative cytometric profile and quantitation of intracellular cytokines (IFN, TNF, IL-2). d, Representative cytometric profile of stemness markers (TCF7, CD27). e, Western blotting confirmation of TCF7 expression. f, Representative cytometric profile of cell proliferation based on Ki67 expression. The profiles were used to generate Fig. 2f. g, Representative CD45RA/CCR7 profile of different CD39 populations. The profiles were used to generate Fig. 2g. (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test).
Supplementary discussion, protocol, figures, tables, methods and references.
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Wang, Z., Ahmed, S., Labib, M. et al. Efficient recovery of potent tumour-infiltrating lymphocytes through quantitative immunomagnetic cell sorting. Nat Biomed Eng 6, 108–117 (2022). https://doi.org/10.1038/s41551-021-00820-y
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