Abstract PO-17: Microphysiologic model of ALK+ anaplastic large cell lymphoma and vascular interactions predicts drug efficacy in a 3D microfluidic chip
Authors: Campisi Marco , Voena Claudia , Mota Ines , Patrucco Enrico , Kamm Roger Dale , Chiarle Roberto 
Published: December 2020

Anaplastic large-cell lymphoma (ALCL) initially responds well to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) therapies (e.g., crizotinib); however, resistance appears once the treatment is concluded and persistent cells are not eradicated. ALCL preferentially grows around blood and lymphatic vessel in the lymph node, and our preliminary data have shown that CCL19/21-CCR7 chemokine-receptor signaling axis might be involved in TKI resistance of ALCL cells. To investigate this mechanism, we developed a microphysiologic model of ALCL interacting with a 3D vasculature using a microfluidic chip. Microfluidic technology has the potential to impact cancer diagnosis and therapy by overcoming the limitations of 2D culture. These ex vivo models are able to uncover the continuous interactions and chemokine signaling that exist between cells of the tumor microenvironment (TME) and evaluate the preclinical efficacy of novel and personalized cancer therapeutics. Two ALK+ ALCL cell lines (COST and Karpas299) were knocked out by CRISPR/Cas9 of the CCR7 receptor, transduced with GFP for microscopic visualization. A commercial microfluidic chip with a central gel channel, flanked by two fluidic media channels, was used to develop the model. A collagen hydrogel (2.5 mg/mL) was injected into the central region of the 3D chip, incubated in sterile humidity chambers, and channels were hydrated with RPMI. 50 µL of 3x106 cells/mL human umbilical vein endothelial cells (HUVECs) suspension was injected twice on the fluidic channel, and the chip was rotated twice to create a confluent hollow-lumen 3D macrovessel. Then, 2x105 cells/mL ALCL cells were added inside the 3D macrovessel. Medium was refreshed daily, supplemented +/- 300 nM crizotinib (ALK TKI). Image analysis was performed using a confocal microscope. The model consisted of a well-formed perfusable macrovessel with ALCL cells flowing inside and interacting with HUVECs. ALCL viability was evaluated with a luminescent readout assay after 3 days of interactions with HUVECs. Strikingly, ALCL cells cultivated within the vasculature showed marked resistance to treatment with ALK TKI compared to cells cultivated in the absence of vessels. CCR7 KO cells showed decreased viability and decreased perivascular localization compared to wild-type control cells. The results suggest that the 3D ALCL-vascular microphysiologic model is a feasible platform with great potential to unveil the molecular mechanisms of drug resistance in a complex TME. The presence of macrovessels was sufficient to induce resistance to ALK TKI, which was decreased in CCR7 KO cells. These data indicate that CCL19/21 and possibly other cytokines produced by vessels generate survival signals in ALCL cells treated with ALK TKI. This physiologically relevant ALCL-vascular model offers a tool to genetically dissect the TME contribution to drug resistance, predict more reliably therapeutic vulnerabilities, and recapitulate patient-specific cell-cell interactions.

Citation Format: Marco Campisi, Claudia Voena, Ines Mota, Enrico Patrucco, Roger Dale Kamm, Roberto Chiarle. Microphysiologic model of ALK+ anaplastic large cell lymphoma and vascular interactions predicts drug efficacy in a 3D microfluidic chip [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-17.