Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics.

Long-range communication between tumor cells and the lymphatic vasculature defines competency for metastasis in different cancer types, particularly in melanoma. Nevertheless, the discovery of selective blockers of lymphovascular niches has been compromised by the paucity of experimental systems for whole-body analyses of tumor progression. Here, we exploit immunocompetent and immunodeficient mouse models for live imaging of Vegfr3-driven neolymphangiogenesis, as a versatile platform for drug screening in vivo. Spatiotemporal analyses of autochthonous melanomas and patient-derived xenografts identified double-stranded RNA mimics (dsRNA nanoplexes) as potent inhibitors of neolymphangiogenesis, metastasis, and post-surgical disease relapse. Mechanistically, dsRNA nanoplexes were found to exert a rapid dual action in tumor cells and in their associated lymphatic vasculature, involving the transcriptional repression of the lymphatic drivers Midkine and Vegfr3, respectively. This suppressive function was mediated by a cell-autonomous type I interferon signaling and was not shared by FDA-approved antimelanoma treatments. These results reveal an alternative strategy for targeting the tumor cell-lymphatic crosstalk and underscore the power of Vegfr3-lymphoreporters for pharmacological testing in otherwise aggressive cancers.The authors thank previous and present colleagues in the CNIO Melanoma Group, particularly Damia Tormo and Lisa Osterloh for help and support at the initial stages of this study; Jose A Esteban (CSIC-UAM) for critical reading of this manuscript; Lionel Larue (INSERM; France) and Martin McMahon (Hunstman Cancer Center, USA) for the Tyr:CreERT2 and BrafCA mouse strains, respectively; and Ignacio Melero at Hospital Clinico, Pamplona, Spain, for Ifnar1-deficient mice. The authors thank Isabel Blanco, Soraya Ruiz, and Virginia Granda (CNIO-Animal Facility Unit), Diego Megias (CNIO-Confocal Unit), and Eduardo Jose Caleiras and Patricia Gonzalez (CNIO-Histopathology Unit) for technical assistance. M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (SAF2017-89533-R), the Asociacion Espanola Contra el Cancer (AECC), Fundacion La Caixa, and an Established Investigator Award by the Melanoma Research Alliance (MRA). D.O. is funded by grants from the Spanish Ministry of Health (AES-PIS PI18/1057) and "Beca Leonardo a Investigadores y Creadores Culturales 2018 de la Fundacion BBVA". The CNIO Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019. S.O. is also supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015-71376-R).S

Recurrent presence of the PLCG1 S345F mutation in nodal peripheral T-cell lymphomas

This work was supported by grants from Asociación Española contra el Cancer (AECC), Ministerio de Economía y Competitividad (MINECO) (SAF2013-47416-R), Instituto Salud Carlos III (ISCIII) – Fondos FEDER, MINECO-AES(RD012/0036/0060, PI10/00621, CP11/00018). RM is supported by the Fundación Conchita Rábago de la Fundación Jiménez Díaz, Madrid (Spain). JG-R is supported by a predoctoral grant from the Fundacion Investigacion Biomedica Puerta de Hierro. Salary support to SG is provided by ISCIII-FEDER (CP11/00018). MS-B is supported by a Miguel Servet contract from ISCIII-FEDER (CP11/00018). The Instituto de Investigación Marqués de Valdecilla (IDIVAL) is partly funded by the Sociedad para el Desarrollo Regional de Cantabria (SODERCAN)

PLCγ1/PKCθ downstream signaling controls cutaneous T-cell lymphoma development and progression

Data de publicació electrònica: 21-10-2021Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLCγ1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLCγ1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKCθ) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKCθ-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKCθ blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKCθ target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKCθ) in 81 human mycosis fungoides samples. In summary, PKCθ can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies.This work has been funded by the Instituto de Salud Carlos III (ISCIII)/FEDER (PI16/00156, PI19/00204, and ASOCIACION LUCHAMOS POR LA VIDA to JPV; PI17/0957 to PLOR). NGD has been supported by a predoctoral contract from UC-IDIVAL. BC holds a RyC contract from MICINN (RYC2018-024004). AEC is funded by ISCIII/MINECO/FEDER (PT17/0009/0019

Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state.

An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8+ T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.We thank the colleagues at the CNIO Melanoma Group, as well as those at the laboratories of H. Peinado and Manuel V. (CNIO), for help and support, I. Blanco, S. Ruiz, V. Granda, S. Rueda (CNIO) and the Animal Facility, Histopathological Unit, Confocal Microscopy Unit and Crystallography and Protein Engineering Unit of CNIO for assistance with the mouse colonies and histopathological and protein analyses, and D. Sancho (CNIC) for the B16-OVAGFP cells and OT-I mouse strain, and for scientific guidance. P. Turko (University of Zurich) provided advice on the statistical analyses of tissue microarrays. We also thank the donors and the Biobank Hospital Universitario Puerta De Hierro Majadahonda (HUPHM)/Instituto De Investigacion Sanitaria Puerta De Hierro-Segovia De Arana (IDIPHISA) (PT17/0015/0020 in the Spanish National Biobanks Network) for the human specimens used in this study. M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (SAF2017-89533-R), Team Science and Established Investigator awards by the Melanoma Research Alliance, and grants from Worldwide Cancer Research and Fundacion 'La Caixa' Health Research 2019. M.S.S., P.O.-R. and J.L.R.-P. are funded by a collaborative grant from the Asociacion Espanola Contra el Cancer (AECC). D.O. is funded by grants from the Spanish Ministry of Health (AES-PIS PI18/1057) and 'Fundacion BBVA-Becas Leonardo a Investigadores y Creadores Culturales 2018'. D.C.-W. was a recipient of a predoctoral fellowship from Fundacion 'La Caixa' and is currently funded by the AECC. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. N.I. and J.M. are funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. M.C.-A. and X.C. were funded by the Immutrain Marie Skodowska-Curie ITN Grant. S.H. received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement numberS

Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis1,2,3. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma4,5. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress6,7. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma8,9,10,11,12,13,14. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear2,14. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches15. Injectable lymphatic tracers have been developed7, but their limited diffusion precludes whole-body imaging at visceral sites16. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive ‘lymphoreporter’17 because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer17,18. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a ‘MetAlert’ discovery platform for drivers and inhibitors of metastasis.M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (project SAF2014-56868-R), the Asociación Española Contra el Cáncer (AECC), the Worldwide Cancer Research, an Established Investigator Award from the Melanoma Research Alliance (MRA), and a L’Oréal Paris USA-MRA Team Science Award for Woman in Scientific Research. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. N.I. and J.M. are funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. J.L.R.-P and P.O.-R are funded by grants FIS 2014/1737, 11/02568 and FIS 2014/01784, 11/1759, respectively, from the Spanish Ministry of Health. F.M. is funded by the AMIT Project/CDTI/CENIT Programme (MICINN), S.O. by SAF2013-44866-R (MINECO), and J.J.B.-C. by an NCI K22CA196750 grant and the TCI Young Scientist Cancer Research Award JJR Fund (P30 CA196521). J.D.M. is the recipient of a postdoctoral fellowship from the ARC Foundation and E.R.-F. from Fundación Científica de la Asociación Española Contra el Cáncer. D.C.-W. is the recipient of a predoctoral fellowship from Fundación La Caixa, and M.C.-A. and X.C. are recipients of the Immutrain Marie Skłodowska-Curie ITN Grant.Peer reviewe