Citation: Pinto, A.T.; Pojo, M.; Rodrigues, R.; Sousa, D.P.; Matthiesen, R.; Carvalho, A.S.; Beck, H.C.; Pires, C.; Eduardo, R.; Pereira, J.S.; et al. SPRY4 as a Potential Mediator of the Anti-Tumoral Role of Macrophages in Anaplastic Thyroid Cancer Cells. Cancers 2023, 15, 4387. https://doi.org/10.3390/ cancers15174387 Academic Editor: David Wong Received: 30 June 2023 Revised: 2 August 2023 Accepted: 18 August 2023 Published: 1 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article SPRY4 as a Potential Mediator of the Anti-Tumoral Role of Macrophages in Anaplastic Thyroid Cancer Cells Ana Teresa Pinto 1,2, * ,† , Marta Pojo 1,† , Ricardo Rodrigues 1,† , Diana Pacheco Sousa 1 , Rune Matthiesen 3 , Ana Sofia Carvalho 3 , Hans C. Beck 4 , Carolina Pires 1 , Rodrigo Eduardo 1 , Joana Simões Pereira 1,5 , Valeriano Leite 1,5,6 and Branca Maria Cavaco 1 1 Unidade de Investigação em Patobiologia Molecular (UIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG), 1099-023 Lisboa, Portugal; marta.pojo@ligacontracancro.pt (M.P.); rmrodrigues@ipolisboa.min-saude.pt (R.R.); diana.sousa@nms.unl.pt (D.P.S.); cs.pires@campus.fct.unl.pt (C.P.); rodrigo.eduardo@research.fchampalimaud.org (R.E.); jpereira@ipolisboa.min-saude.pt (J.S.P.);vleite@ipolisboa.min-saude.pt (V.L.); bcavaco@ipolisboa.min-saude.pt (B.M.C.) 2 Instituto de Biomedicina (iBiMED), Universidade de Aveiro, 3810-193 Aveiro, Portugal 3 NMS Research, NOVA Medical School, Faculdade de Ciências Médicas (NMS|FCM), Universidade Nova de Lisboa, 1169-056 Lisboa, Portugal; rune.matthiesen@nms.unl.pt (R.M.); ana.carvalho@nms.unl.pt (A.S.C.) 4 Centre for Clinical Proteomics, Department of Clinical Biochemistry, Odense University Hospital, DK-5000 Odense, Denmark; hcbeck@health.sdu.dk 5 Serviço de Endocrinologia, IPOLFG, 1099-023 Lisboa, Portugal 6 NOVA Medical School, Faculdade de Ciências Médicas (NMS|FCM), Universidade Nova de Lisboa, 1169-056 Lisboa, Portugal * Correspondence: anapinto@ua.pt † These authors contributed equally to this work. Simple Summary: Anaplastic thyroid cancer (ATC) displays a high density of tumor-associated macrophages, which modulate invasiveness and tumor growth. However, the molecular mecha- nisms underlying the communication between tumor cells and macrophages in ATC tumor mass are still poorly understood. Our study observed bidirectional communication mechanisms between macrophages and ATC cells, in which macrophages influenced cancer cell viability and invasive phenotype and cancer cells modulated macrophage polarization. We identified SPRY4 as a crucial me- diator in this interplay, being a candidate tumor suppressor gene in this context, which may be useful to disclose new processes related to ATC aggressiveness that can lead to future therapeutic options. Abstract: Anaplastic thyroid carcinoma (ATC) is the most lethal subtype of thyroid cancer, with high invasive and metastatic potential, not responding to conventional treatments. Its aggressiveness may be influenced by macrophages, which are abundant cells in the tumor microenvironment. To investigate the role of macrophages in ATC aggressiveness, indirect co-cultures were established between ATC cell lines and THP-1-derived macrophages. Macrophages significantly increased both the migration and invasion of T235 cells (p < 0.01; p < 0.01), contrasting with a decrease in C3948 (p < 0.001; p < 0.05), with mild effects in T238 migration (p < 0.01) and C643 invasion (p < 0.05). Flow cytometry showed upregulation of CD80 (pro-inflammatory, anti-tumoral) and downregulation of CD163 (anti-inflammatory, pro-tumoral) in macrophages from co-culture with T235 (p < 0.05) and C3948 (p < 0.05), respectively. Accordingly, we found an upregulation of secreted pro-inflammatory mediators (e.g., GM-CSF, IL-1α; p < 0.05) in C3948–macrophage co-cultures. Proteomic analysis showed the upregulation of SPRY4, an inhibitor of the MAPK pathway, in C3948 cells from co-culture. SPRY4 silencing promoted cancer cell invasion, reverting the reduced invasion of C3948 caused by macrophages. Our findings support that macrophages play a role in ATC cell aggressiveness. SPRY4 is a possible modulator of macrophage–ATC cell communication, with a tumor suppressor role relevant for therapeutic purposes. Keywords: anaplastic thyroid cancer; macrophages; invasion; migration; SPRY4; anti-tumoral Cancers 2023, 15, 4387. https://doi.org/10.3390/cancers15174387 https://www.mdpi.com/journal/cancers