Abstract Background: Head and neck squamous carcinoma (HNSCC) results in poor patient outcomes driven primarily by locoregional and distant metastatic tumor spread. We have demonstrated that highly phosphorylated/active MAPKAPK-2 (MK2) is a poor prognostic factor in HNSCC patients. MK2 is associated with the expression of pro-inflammatory cytokines that are linked with creating a pro-tumorigenic microenvironment. Nevertheless, how MK2 contributes to HNSCC development has not been established. In this study, we aimed to identify the effect of MK2 on cancer progression, immune infiltration, and cytokine secretion. Method: To assess the effect of tumor MK2 in vivo, we used CRISPR-Cas9 to knock out (KO) MK2 in a metastatic murine HNSCC cell line, Ly2, and orthotopically implanted wildtype (WT) or MK2 KO cells into the floor-of-mouth of Balb/c mice. At the endpoint of the experiment, the tumors were excised, and myeloid and lymphocyte immune profiling was done via flow cytometry. Mouse lungs and cervical lymph nodes were dissected, paraffin-fixed, sectioned, and H&E stained, and the presence/quantity of metastatic foci was evaluated by a blinded board-certified pathologist. We broadly evaluated the tumor secretome via a 105-cytokine dot blot array using 8 mg of the same resected tumor sample and performed densitometric analysis comparing WT and KO tumors. Results: Loss of MK2 significantly reduced primary tumor growth, as well as lymph node and lung metastasis, compared to WT tumors. Tumor immunophenotyping by flow cytometry revealed that MK2 KO tumors had increased overall immune cell (CD45+) infiltration, but decreased quantities of immunosuppressive neutrophils (CD11b+Ly6G+Arg1+Ly6C-) and a shift in macrophage polarity favoring M1 (anti-tumor) macrophages (CD11b+Ly6G-F4-80+CD80+). In addition, there were increased quantities of anti-tumor immune cells including natural killer (NK)-T, CD4+, and CD8+ T cells in the MK2-KO tumors, while the proportion of CD4+CD25+ Tregs and exhausted CD8 T cells (CD8+PD1+) was decreased compared to WT tumors. Furthermore, cytokine array analysis revealed that secreted levels of several cytokines, including CXCL1, CXCL2, GM-CSF, G-CSF, IL-1alpha, and IL-6, were significantly lower in MK2 KO tumors compared to WT tumors. Conclusion: These results show that tumoral MK2 signaling promotes tumor growth, metastasis, immunosuppressive inflammation, and pro-tumorigenic cytokine release which substantiates future therapeutic targeting of this pathway. Citation Format: Dakota D.D. Okwuone, Deri Morgan, Alyssa Schmidt, Devin Shrock, Yuting Lin, Hao Gao, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in head and neck squamous cell carcinoma [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 C010.

Abstract Disseminated cancer cells (DCCs) arriving to a new organ may either proliferate and form metastasis, get eliminated by immune cells, or enter a quiescent stage. The factors that determine the DCCs’ fate, including getting quiescent DCCs to re-initiate proliferation and successfully evade the immune system, remain poorly understood, in part due to the lack of robust mouse models. We established a pancreatic ductal adenocarcinoma (PDAC) mouse model of dormant DCCs in the liver. An “immunization protocol” was used to mimic PDAC resection in patients: KPC1199 PDAC cells were injected to establish primary tumors and allow an adaptive immune response to develop against the cancer cells; the tumors were surgically resected; and new KPC1199 cells were intrasplenically injected to establish experimental liver metastases. The overwhelming majority of the KPC1199 cells were eliminated. No metastases spontaneously formed during 20 months of observation, but about 3-8 sporadic, single DCCs were found per liver cross section, and 94% of these DCCs were negative for proliferation markers. Although the model is based on entraining an adaptive immune response, T cell depletion resulted in only 25-30% of mice developing just 1-3 metastases each. This suggests that T cell depletion results in metastasis from the small percentage of DCCs that were proliferating. The DCC-hosting mice therefore represents of model to identify factors that can trigger further DCC proliferation and metastatic recurrence. Patients with PDAC often experience significant stress, leading to excess of endogenous glucocorticoids (GCs). Additionally, synthetic GCs are used during e.g., chemotherapy to manage side effects. To mimic elevated GC levels in patients, we treated DCC-hosting mice with synthetic GCs resulting in an increased percentage of proliferating DCCs (increasing from <6% of the examined cells in control mice to about 36%). Yet, no metastases formed. GC treatment also decreased T cell and increased neutrophil liver infiltration. Neutrophils can form protease-containing neutrophil extracellular traps (NETs), which we previously showed trigger DCC proliferation by proteolytic remodeling of the extracellular matrix protein laminin. We found that neutrophils from GC-treated mice spontaneously formed more NETs, and additionally, NET-generated laminin remodeling was observed in the liver metastasis. Together, the data suggest, that GC treatment drive quiescent DCCs to proliferate via neutrophils but alone this is insufficient to cause metastases. Consistent with this interpretation, T cell depletion combined with GC treatment resulted in multiple metastases in all mice examined. Of note, deleting the GC receptor in the DCCs did not reduce metastases, supporting that GCs act on the host and not the cancer cells. In conclusion, our data suggest that for quiescent DCCs to form metastases, they need signals that trigger proliferation and simultaneously, they must overcome immune surveillance. Elevated GC levels or GC treatment in immune suppressed individuals represents such a scenario. Citation Format: Xiao Han, Xueyan He, Yuan Gao, Sicheng Pan, Mikala Egeblad. Glucocorticoids establish a metastatic-promoting tumor microenvironment in a PDAC mouse model [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 C004.

Abstract Immune checkpoint inhibitors (ICIs) have emerged as a significant advance in the treatment of various cancer types, including breast cancer, though their efficacy varies widely depending on the cancer subtype and the stage of the disease. Following their initial approval in metastatic triple-negative breast cancer (TNBC), ICIs have shown promising results in other contexts, including early-stage cancers and ER+ and HER2+ subtypes. However, even in advanced TNBC the objective response rate to monotherapy ICI is only ∼20%. These results highlight the need to better understand determinants of ICI response in breast cancer. A detailed, high-resolution definition of the tumor immune microenvironment (TIME) will be critical for predicting and engineering effective responses to ICIs. In previously published work, we and others found that the antioxidant transcription factor NRF2 is constitutively active in breast cancer and is associated with tumor recurrence, metastasis, and poor prognosis. However, the mechanisms through which NRF2 promotes breast cancer progression remain unclear. In the current study, we explore the role of NRF2 in reshaping the TIME and its impact on breast cancer progression using preclinical models, flow cytometry, and transcriptomic analyses. We find that NRF2 is constitutively activated in a subset of human and mouse breast cancers where it functions to suppress inflammatory gene expression. In models of recurrent and metastatic mammary tumors, NRF2 knockdown results in a marked reduction in tumor growth rates. Furthermore, the NRF2 inhibition significantly alters the composition of innate and adaptive immune cell populations within the tumor microenvironment. Cytokine profiling revealed that these immune alterations are associated with increased levels of inflammatory cytokines in NRF2-deficient tumor cells compared to controls. RNA sequencing of tumor cells further supports the observed shifts in the immune landscape associated with NRF2 deficiency. Collectively, our findings demonstrate that the loss of NRF2 impairs tumor growth in part through remodeling the TIME and suggest that NRF2 may be a promising therapeutic target for sensitizing breast cancers to ICIs. Citation Format: Yasemin Ceyhan Ozdemir, James V. Alvarez. NRF2 regulation of the immune microenvironment in breast cancer [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 B014.

Abstract Solid tumors are innervated by distinct branches of the peripheral nervous system, and increased tumor innervation has been associated with poor cancer outcomes. However, it remains unexplored whether different types of nerves function together to sense and respond to a tumor, and how neural networks shape cancer immunity. As a major interoceptive system, vagal nerves sense a large variety of body signals and are critical for maintaining physiological homeostasis of visceral organs including the lung. While distinct vagal sensory neuron (VSN) subtypes have been identified to differently regulate pulmonary functions, it is unknown whether these VSNs interact with lung tumors and how they influence cancer progression. Here, we uncover that sensory nerves act through the sympathetic circuit to restrain anti-cancer immunity. Mechanistically, our data suggest that lung tumors hijack the vagal interoceptive system to activate sympathetic efferent nerves in the tumor microenvironment (TME), which in turn drive immune suppression via β2-adrenergic signaling in alveolar macrophages. Our key findings include: By applying anterograde labeling, tissue-clearing and 3D imaging to genetically engineered mouse models, we found that lung adenocarcinoma is richly innervated by vagal sensory nerves. We also showed that neurotrophic factors produced by cancer cells directly promote vagal sensory innervation. Furthermore, scRNA-seq analysis revealed tumor-induced transcriptional reprogramming of lung-innervating VSNs. Using multiple genetic tools to selectively label and deplete different VSN subtypes, we found that Npy2r + /Trpv1 + VSNs but not P2ry1 + VSNs innervate lung tumors and control lung cancer progression by inhibiting anti- cancer immunity. At the organism level, we demonstrated that vagal NPY2R/TRPV1 neurons inhibit anti-cancer immunity via the sympathetic axis. Depletion of vagal NPY2R/TRPV1 neurons resulted in reduced sympathetic nerve activity and a decreased norepinephrine level in the lung TME, whereas pharmacological activation of the sympathetic pathway suppressed the anti-tumor immune responses and restored tumor growth in mice lacking Npy2r + /Trpv1 + VSNs. At the molecular and cellular level, we identified that the vagal-to-sympathetic axis predominantly functions through β2-adrenergic signaling in alveolar macrophages (AMs) to drive immune suppression. AM depletion or selective deletion of β2-adrenergic receptor (ADRB2) in AMs derepressed the anti-tumor immunity and abolished the tumor-inhibiting effect of Npy2r + /Trpv1 + VSN ablation. Taken together, our study establishes an essential role of the sensory-to-sympathetic circuit in controlling cancer immunosurveillance, supporting the notion that the functional integration of sensory and sympathetic nerves systemically regulates anti- cancer immunity. Translationally, our findings from the preclinical model suggest that targeted disruption of the vagal sensory-to-sympathetic axis may provide new treatments for visceral organ cancers by enhancing anti-tumor immunity. Citation Format: Haohan Wei, Chuyue Yu, Bo Hu, Xing Zeng, Hiroshi Ichise, Ronald N. Germain, Rui Chang, Chengcheng Jin. A vagal sensory-to-sympathetic axis restrains anti-tumor immunity [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 PR013.

Abstract Breast cancer ranks as the most commonly diagnosed cancer in women with 90% of associated deaths caused by distant metastasis. The metastatic lesions in brain, lung, liver, bone and lymph nodes in human breast cancer are not arbitrary, but rather are a complex mechanism of extrinsic and intrinsic regulatory factors controlling organ-specific colonization. Specialized mechanisms driving bone, brain and lung cancer have been described. However, the studies of liver and lymph node metastatic drivers are limited despite the prevalence of metastases in these organs. A significant limitation is the lack of genetically engineered mouse models that develop mammary tumors that spontaneously metastasize to liver and lymph nodes. Our previous research identified the role for E2F5 in both mammary gland development and breast cancer. Loss of E2F5 resulted in tumorigenesis after a latency of one year and 70% of mice showed metastatic lesions in lung, lymph node and liver. Transplantation of these spontaneous tumors into FVB MMTV Cre mice recapitulated the metastatic pattern observed in the primary tumors. We propose that E2F5 regulates drivers of metastasis and potentiates organ tropism to lymph nodes and the liver. To address this hypothesis, we conducted serial transplantations of tumors originating from the lymph nodes and liver back into the mammary gland. This enrichment study led to a significant increase in organ-specific metastasis, with 60% and 77% of penetrance observed in two distinct mouse lineages with liver (LVM) and lymph node tropism (LNM). Immunohistochemical studies confirmed a complete epithelial-mesenchymal transition (EMT) phenotype in LVM that is conserved in the cell lines generated. LNM showed a partial EMT with a more epithelial phenotype in cell lines. We observed the presence of an immune enriched environment in primary and metastatic tumors in both lineages. Bulk-RNA sequencing studies uncovered the presence of unique organotropic pathways associated with liver and lymph node metastasis. We observed enriched pathways from the primary tumor as major component of our organotropic drivers with a clear distinction between liver and lymph node enriched lineages. Additionally, a gradual acquisition of distant organ-specific markers and signaling pathways suggests that phenotypic mimicry and tumor plasticity are key for successful events of colonization to liver and lymph nodes in our model. With these results, we unravel potential mechanisms responsible for the organotropic metastases in breast cancer under the absence of the transcription factor E2F5. Citation Format: Jesus A. Garcia Lerena, Briana To, Jing-Ru Jhan, Eran Andrechek. E2F5 conditional knockout in mammary epithelium drives organotropic metastasis in breast cancer [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 A004.

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer (BC) cases worldwide, yet due to its aggressive phenotype and lack of targeted therapies it has the worst prognosis amongst BC subtypes. Up to 40% of TNBC patients will relapse after treatment and develop distant metastasis. Metastasis is the complex process by which disseminated tumor cells (DTCs) leave the primary tumor and grow at distal sites. Since most metastases occur within 3 years of diagnosis, the rapid onset and lack of effective treatments in metastatic TNBC (mTNBC) means less than 20% of patients survive after 4 years. Even though it is the most “immune-activated” BC subtype, and immune checkpoint blockade (ICB) has shown clinical benefits in early TNBC, there is a clear need for novel immunotherapeutic approaches for patients with mTNBC. Natural killer (NK) cells are innate lymphocytes that function as the body’s first line defense against metastatic cancer cells. NK cells eliminate DTCs in the blood or at distal sites via NK cell cytotoxicity (NKCC) and this process is known to be impaired in successful metastasis. We hypothesized that other cells within the tumor microenvironment (TME) can inhibit NKCC at the earliest stages of metastatic outgrowth. We have used Cherry-niche, an in vivo labelling tool, to characterize how TME cells change in early and established lung micro-metastases by single-cell RNA sequencing (scRNA-seq) and flow cytometry. Our initial analyses showed that monocytes are highly enriched, not only in the entire metastatic lung, but specifically in the established niche surrounding DTCs. Although we detected a slight increase in total NK cells, there was a shift in maturation status with fewer mature NK cells (CD27-CD11b+) and more immature NK cells (CD27+/-CD11b-) in the metastatic lung. Furthermore, NK cells were largely absent from the metastatic niche and those that were present displayed a dysfunctional phenotype. CellChat and NicheNet analyses of our scRNA-seq data predicted that several receptor-ligand interactions, including the cytokine macrophage migration inhibitory factor (MIF), can mediate the crosstalk between NK cells and monocytes. Next, we developed an in vitro 3D co-culture system to study the effect of blood-derived monocytes on human NKCC against mTNBC cells. We found that monocytes can suppress NKCC against MDA-MB-231 cells and decrease the viability of mature NK cells. Furthermore, recombinant MIF was able to directly inhibit NKCC, whilst a selective MIF antagonist was able to rescue monocyte-induced impairment of NKCC. These data implicate MIF as a critical monocyte-derived factor in the suppression of NK cell function against mTNBC cells. Future work will aim to identify the exact mechanism by which MIF inhibits NKCC, whilst also evaluate the effect of monocyte depletion and MIF inhibition on metastatic outgrowth and NK cell activation/maturation in vivo. This study will reveal novel inter-cellular dynamics occurring in the metastatic lung, as well as immunotherapeutic targets that may restore NKCC in mTNBC. Citation Format: Christos Ermogenous, Luigi Ombrato, Gordon Beattie. Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer [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 B022.

Abstract Triple-negative breast cancer (TNBC) is associated with a high risk of distant metastasis, particularly to the lung and liver. Both intrinsic characteristics of cancer cells and the tumor microenvironment (TME) influence TNBC growth and metastasis. The TME, comprising stromal, immune, and endothelial cells, exhibits heterogeneity both between and within cell types. Identifying the molecular characteristics of TME cells that affect cancer progression is crucial. We aim to investigate this using single-cell RNA sequencing of patient-derived xenograft (PDX) models to explore TME cells and genes involved in TNBC metastasis. We established PDX models by transplanting tumor tissues from 26 TNBC patients into immunodeficient mice. These models were categorized based on their metastatic potential and patient outcomes: non-metastatic (n=5) and metastatic (n=4). Single-cell RNA sequencing of nine PDX tumors revealed significant differences in gene expression of murine stromal cells between metastatic and non-metastatic models. We identified ten genes (Serpinb2, Spp1, Tnc, Thbs1, Timp1, Il11, Mt2, Crabp1, Cck, Mt1) that were highly expressed in the stromal cells of metastatic PDX models. To test if TNBC cells influence these fibroblast genes, we exposed NIH3T3 fibroblasts to conditioned media from the 4T1 TNBC cell line. In three independent experiments, only Crabp1 expression in NIH3T3 cells was consistently increased by 4T1- conditioned media. CRABP1, a retinoic acid-binding protein, is known for its role in differentiation and proliferation by regulating MAPK signaling. Although Crabp1-positive cancer-associated fibroblasts have been noted, its role in TNBC metastasis was previously unexplored. We investigated CRABP1 function using a CRABP1 knockdown NIH3T3 cell line. CRABP1 knockdown led to reduced invasion and migration of 4T1 cells in trans-well assays and decreased invasiveness of 4T1 spheroids in a collagen matrix. Additionally, CRABP1 knockdown NIH3T3 cells showed reduced proliferation in vitro and fewer alpha-SMA positive cancer-associated fibroblasts in co-injected 4T1 tumors in BALB/C mice. In summary, CRABP1, identified through single-cell RNA sequencing of stromal cells in TNBC PDX models, is a potential regulator of TNBC metastasis, affecting cancer cell migration, invasion, and fibroblast proliferation. Citation Format: Woohang Heo, Yujeong Her, Sieun Yang, Dakyung Lee, Rokhyun Kim, Jong-Il Kim, Hyeong-Gon Moon. Single cell RNA sequencing of triple negative breast cancer patient-derived xenograft model identifies CRABP1 of cancer-associated fibroblast as a key regulator of breast cancer metastasis [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 A034.

Abstract There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear; while biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the tumor-surrounding microenvironment, contextually in aging, has been overlooked. Dermal fibroblasts are known to have profound impact on melanoma progression. We examined if both sex-dependent and age-related changes in these fibroblasts can alter the course of melanoma tumor growth, visceral metastasis, and responses to therapy. We find that skin fibroblasts undergo age-mediated sex-differential changes in their proliferation, senescence, ROS production and stress response. We find that aged male fibroblasts drive an invasive, therapy-resistant phenotype in melanoma cells and promote metastasis in aged male mice by increasing AXL expression. Intrinsic aging promotes sex- dimorphic BMP2 secretion exclusively by aged male fibroblasts, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of invasive phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. Our data provide an integrated view of how host factors such as advancing age and biological sex alter the tumor microenvironment and contribute to disease progression. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy and achieve gender-equitable healthcare. Citation Format: Yash Chhabra, Ashani T. Weeraratna. Sex-dependent changes in the aged melanoma tumor microenvironment influence metastasis and therapeutic responses [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 PR003.

Abstract Metastasis is the leading cause of breast cancer (BC) death, and tumor cells must migrate and invade to metastasize. BC cells that express the actin regulatory protein MenaINV have an enhanced ability to migrate and intravasate within the primary tumor, and extravasate at secondary sites. Though chemotherapy improves patient survival, treatment with paclitaxel, a chemotherapy used for BC, leads to upregulation of MenaINV and an increase in metastasis in mice. MenaINV expression can be induced in BC cells through Notch1 signaling with macrophages, which are often recruited to tumors in response to chemotherapy. MenaINV-expressing cells are also resistant to paclitaxel, begging the question of whether paclitaxel increases MenaINV by de novo induction or by selectively killing non-MenaINV-expressing cells. We hypothesized that paclitaxel causes de novo MenaINV induction by increasing macrophage-tumor cell Notch signaling. Understanding this pro-metastatic effect of chemotherapy is crucial to refining treatment strategies. MMTV-PyMT mice bearing spontaneous mammary tumors were used as a model of BC. Mice were treated with paclitaxel or vehicle every 5 days for a total of 3 treatments. To study the role of macrophages in the paclitaxel-mediated MenaINV increase, in addition to paclitaxel, one cohort of mice was co-administered the macrophage-depleting agent clodronate or vehicle. To study the role of Notch signaling, a second cohort of mice was co-administered the Notch inhibitor DAPT or vehicle. Immunofluorescence staining was used to evaluate tumors for: %MenaINV+ area, proportion of apoptotic cells, macrophage density, and macrophage expression of Notch ligands Jag1 and Jag2. Paclitaxel-treated tumors expressed significantly more MenaINV even when tumor cell death did not increase, indicating that chemotherapy increases MenaINV expression by induction. Both macrophages and Notch signaling were needed for this effect to occur. Interestingly, paclitaxel did not increase macrophage infiltration or macrophage expression of Notch ligands, suggesting that paclitaxel may increase MenaINV by acting on tumor cells to promote Notch signaling. Paclitaxel causes de novo MenaINV induction that is macrophage- and Notch-dependent. Studies aiming to further elucidate the mechanism behind paclitaxel-mediated MenaINV induction are ongoing. These results lay the groundwork for novel microenvironment-based therapies to alleviate the pro-metastatic effects of chemotherapy in BC. Citation Format: Madeline Friedman-DeLuca, George S. Karagiannis, Camille L. Duran, Luis Rivera Sanchez, Prachiben P. Patel, Suryansh Shukla, Nicole D. Barth, Ved P. Sharma, Michael Papanicolaou, John S. Condeelis, Maja H. Oktay, David Entenberg. Chemotherapy causes de novo induction of invasive breast cancer cells [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 C037.

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.