Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVβ3, αVβ5, αVβ6, αVβ8, and α5β1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 β-subunit partners: β6 = β8 > β3 > β1 = β5.

Solid tumors, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. We found that chemokine-like factor ( CKLF ) is highly expressed by various solid tumor cells and transcriptionally up-regulated by MYCN. Using the MYCN-driven high-risk neuroblastoma as a model system, we demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing CD4 + cells, inducing immunosuppression and tumor aggression. Genetic depletion of CD4 + T regulatory cells abolishes the immunorestrictive and protumorigenic effects of CKLF. Our work supports that disrupting CKLF-mediated cross-talk between tumor and CD4 + suppressor cells represents a promising immunotherapeutic approach to battling MYCN-driven tumors.

Abstract Neuroblastoma, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. Here we combined analyses of human neuroblastoma with live tracking and functional studies of the tumor microenvironment (TME) in zebrafish. We identified chemokine-like factor (CKLF) as a key driver of MYCN-mediated immunosuppression and neuroblastoma aggression. We showed that human MYCN-activated neuroblastoma upregulates and secretes CKLF, which is a reliable predictor of poor patient survival. Analysis of primary patient samples demonstrates a strong positive association between MYCN and CKLF expression in neuroblastoma cells together with the enrichment of FOXP3+ T cells in the TME. Taking advantage of in vivo zebrafish models of MYCN-driven neuroblastoma that resemble human high-risk disease, we demonstrated that cklf overexpression in zebrafish neural crest cells induces an immunosuppressive TME while promoting rapid tumor onset and progression. We also demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing Cd4+ cells to induce immunosuppression and tumor aggression. Conversely, genetic depletion of cklf in tumor cells reduces the recruitment of Cd4+ cells to the TME while increasing cytotoxic Cd8+ and natural killer cells infiltration, inhibiting neuroblastoma development in zebrafish. Our work provides the first example that MYCN can activate CKLF to allure CD4+ immune cells to the TME and incite immunosuppression, positioning CKLF as a potential novel immunotherapeutic target for the treatment of MYCN-driven high-risk neuroblastoma. Citation Format: Xiaodan Qin, Hui Feng, Andrew Lam, Xu Zhang, Satyaki Sengupta, Bryan Iorgulescu, Sanjukta Das, Zhenwei Zhou, Tao Zuo, Grace Meara, Madison Rager, Alexander Floru, Hongru Ni, Chinyere Kemet, Divya Veerapaneni, Daniel Kashy, Liang Lin, Kenneth Lloyd, Lauren Kwok, Kaylee Smith, Raghavendar Nagaraju, Rob Meijers, Craig Ceol, Ching-Ti Liu, Sanda Alexandrescu, Catherine J. Wu, Derin Keskin, Rani George. CKLF attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C002.

Cell-surface receptors can be difficult to express and purify for structural and biochemical studies due to low expression levels, misfolding, aggregation, and instability. Cell-surface receptor ectodomains are more amenable to large-scale production, but this requires designing and testing various truncation constructs. However, since each protein is unique, testing these constructs individually for many targets is a time-consuming process. In this context, we present a high-throughput ELISA fluorescence approach that allows the rapid assessment of numerous recombinant constructs simultaneously. Cell-surface ectodomains are expressed in small scale, enzymatically biotinylated, and detected using a C-terminal His-tag. As an example, we tested the expression of truncation constructs for the neurexin, neuroligin, and latrophilin families and show that the small-scale ELISA allowed us to prioritize well-expressing construct for large-scale production. By employing this method, one can efficiently detect clones with low expression levels, streamlining the process and saving valuable time in identifying optimal candidates for further study.

Abstract Neuroblastoma, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. Here we combined analyses of human neuroblastoma with live tracking and functional studies of the tumor microenvironment (TME) in zebrafish. We identified a tumor-associated chemokine (TAC) as a key driver of MYCN-mediated immunosuppression and neuroblastoma aggression. Taking advantage of in vivo zebrafish models of MYCN-driven neuroblastoma that resemble human high-risk disease, we demonstrated that TAC overexpression in zebrafish neural crest cells induces an immunosuppressive TME while promoting rapid tumor onset and progression. We also demonstrated that as early as the premalignant stage, tumor cells secrete TAC to attract CCR4-expressing Cd4+ cells to induce immunosuppression and tumor aggression. In addition, genetically depleting Cd4+ T regulatory cells abolishes the immunorestrictive and pro-tumorigenic effects of TAC. We showed that human MYCN-activated neuroblastoma upregulates and secretes TAC, which is a reliable predictor of poor patient survival. Analysis of primary patient samples demonstrates a strong positive association between MYCN and TAC expression in neuroblastoma cells together with the enrichment of FOXP3+ T cells in the TME. Our work provides the first example that MYCN can activate TAC to allure CD4+ immune cells to the TME and incite immunosuppression, positioning TAC as a potential novel immunotherapeutic target for the treatment of MYCN-driven high-risk neuroblastoma. Citation Format: Xiaodan Qin, Andrew Lam, Xu Zhang, Satyaki Sengupta, J. Bryan Iorgulescu, Sanjukta Das, Zhenwei Zhou, Tao Zuo, Grace K. Meara, Madison Rager, Alexander E. Floru, Hongru Ni, Chinyere Kemet, Divya Veerapaneni, Daniel Kashy, Liang Lin, Kenneth Lloyd, Lauren Kwok, Kaylee S. Smith, Raghavendar T. Nagaraju, Rob Meijers, Craig Ceol, Ching-Ti Liu, Sanda Alexandrescu, Catherine J. Wu, Derin B. Keskin, Rani E. George, Hui Feng. A tumor-associated chemokine attracts CCR4-expressing CD4+ cells to foster immune repression and tumor aggressiveness in MYCN-driven neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C20.

Solid tumors, especially those with aberrant MYCN activation, harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance1,2, yet the underlying mechanisms are elusive and effective strategies to tackle this challenge are lacking. Here we demonstrated the crucial role of T regulatory (Treg) cells in MYCN-mediated immune repression and tumor aggression using high-risk neuroblastoma (NB) as a model system. Human MYCN-activated NB attracts CD4+ Treg cells, which are also found enriched in MYCN-high primary patient samples. Zebrafish MYCN-overexpressing neural crests recruit Cd4+ cells before tumor formation and induce an immunosuppressive microenvironment, thereby promoting tumor onset and progression. Strikingly, disruption of Treg cells through depletion of forkhead box protein 3a restores anti-tumor immunity and impairs NB development. Together, our studies establish Treg cells as the key driver of MYCN-mediated immunosuppression and tumor aggressiveness, providing mechanistic insights and therapeutic implications.