Primary Tumor Research Articles

Primary Results of NRG-RTOG1106/ECOG-ACRIN 6697: A Randomized Phase II Trial of Individualized Adaptive (chemo)Radiotherapy Using Midtreatment 18F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography in Stage III Non-Small Cell Lung Cancer.

NRG-RTOG0617 demonstrated a detrimental effect of uniform high-dose radiation in stage III non-small cell lung cancer. NRG-RTOG1106/ECOG-ACRIN6697 (ClinicalTrials.gov identifier: NCT01507428), a randomized phase II trial, studied whether midtreatment 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) can guide individualized/adaptive dose-intensified radiotherapy (RT) to improve and predict outcomes in patients with this disease. Patients fit for concurrent chemoradiation were randomly assigned (1:2) to standard (60 Gy/30 fractions) or FDG-PET-guided adaptive treatment, stratified by substage, primary tumor size, and histology. All patients had midtreatment FDG-PET/CT; adaptive arm patients had an individualized, intensified boost RT dose to residual metabolically active areas. The primary therapeutic end point was 2-year centrally reviewed freedom from local-regional progression (FFLP), defined as no progression in or near the planning target volume and/or regional nodes. FFLP was analyzed on a modified intent-to-treat population at a one-sided Z-test significance level of 0.15. The primary imaging end point was centrally reviewed change in SUVpeak from baseline to midtreatment; its association with FFLP was assessed using the two-sided Wald test on the basis of Cox regression. Of 138 patients enrolled, 127 were eligible. Adaptive-arm patients received a mean 71 Gy in 30 fractions, with mean lung dose 17.9 Gy. There was no significant difference in centrally reviewed 2-year FFLP (59.5% and 54.6% in standard and adaptive arms; P = .66). There were no significant differences in protocol-specified grade 3 toxicities, survival, or progression-free survival (P > .4). Median SUVpeak and metabolic tumor volume (MTV) in the adaptive arm decreased 49% and 54%, from pre-RT to mid-RT PET. However, ΔSUVpeak and ΔMTV were not associated with FFLP (hazard ratios, 0.997; P = .395 and .461). Midtreatment PET-adapted RT dose escalation as given in this study was safe and feasible but did not improve efficacy outcomes.

Predicting pan-cancer immune-checkpoint therapy and prognosis with a chromatin-accessibility-related alternative splicing signature: a retrospective study

Background: This study examines alternative splicing (AS) events in genes linked to chromatin accessibility in various cancers and their relation to the tumor immune microenvironment. Methods: Data from the Cancer Genome Atlas Database (TCGA) were used to identify independent prognostic factors for pan-cancer. We explored the correlation between differentially expressed genes and tumor immunity, including immune checkpoint genes, tumor development, and immune cells. A regulatory network diagram of alternative splicing-splicing factors (AS-SFs) was constructed to find potential immunotherapy targets. Results: IRF5 and E2F8 genes showed significant differential expression in pan-cancer. Age, cancer grade, primary tumor, cancer lymph nodes, and distant metastasis were independent prognostic factors. The risk model achieved good predictive performance, with AUC values of 0.705, 0.746, 0.743, and 0.743 for 1-year, 3-year, 5-year, and 10-year survival predictions, respectively. Positive correlations were found between IRF5/E2F8 and CD274/CTLA4 in certain cancers using TIMER and CIBERSORT software. Conclusions: AS events in chromatin accessibility genes (IRF5 and E2F8) have significant predictive value in pan-cancer prognosis. Our model assesses patient survival probability and highlights the synergistic impact of immune checkpoints and the AS-SF regulatory network on tumor immunotherapy.

Solitary fibrous tumor of the gallbladder: a case report

Abstract Background Primary solitary fibrous tumors (SFTs) of the gallbladder are rare. Here, we report the case of a patient who underwent surgical treatment for a primary SFT originating in the gallbladder. Case presentation A 48-mm gallbladder tumor was detected in a 70-year-old man using abdominal ultrasonography at a primary hospital, and he was subsequently referred to our department. A 50-mm enhanced tumor in the gallbladder was identified using computed tomography. Magnetic resonance imaging revealed a smooth-marginated tumor with hyperintensity on T2-weighted imaging. 18F-Fluorodeoxyglucose positron emission tomography confirmed high-level fluorodeoxyglucose uptake in the gallbladder tumor in the early phase without increasing uptake in the later phase. Surgical resection was planned to evaluate the tumor diagnosis. Initially, we performed open cholecystectomy with wedge resection of the gallbladder bed. Intraoperative pathological examination suggested gallbladder cancer; therefore, we performed radical surgery, including resection of the common bile duct, extended radical lymphadenectomy, and choledochojejunostomy. Ultimately, the final pathological examination revealed an SFT originating from the gallbladder with a negative surgical margin. Postoperatively, the patient developed bile leakage that was treated with tube drainage. The patient recovered satisfactorily and was discharged on postoperative day 20. At 24 months postoperatively, the patient was in good general condition without recurrence. Conclusions We report a rare case of a primary SFT originating in the gallbladder. Clinicians should be aware that SFT can be found in the gallbladder, and when it is difficult to make a preoperative diagnosis, surgical treatment should be considered.

Abstract B025: Primary and metastatic tumors from triple negative breast cancer possess distinct immunological profiles

Abstract Metastatic triple negative breast cancer (mTNBC) is almost always fatal due to progressive chemoresistance and a lack of therapeutic targets. Stromal cells, including tumor-infiltrating lymphocytes (TILs) and cancer associated fibroblasts (CAFs), are linked to differences in prognosis and therapy response. While evidence in primary TNBC demonstrates how TIL characteristics may influence outcomes and treatment efficacy, studies in the metastatic setting are lacking. Here, we aimed to characterize TIL phenotype and localization relative to CAFs and cancer cells between primary and metastatic tumors in mTNBC, and to correlate these features with clinical outcomes. CAF cellularity was assessed in H&E stained sections of 15 primary and 40 metastatic lesions, including brain (n=4), liver (n=5), lymph node (n=3), chest wall (n=4), as well as local (n=12) and distant cutaneous (n=12) metastases. Based on the surface area covered by fibroblasts, CAF cellularity was designated as low (0-33%) or high (34-100%). Immunohistochemical staining (IHC) was performed to identify cytotoxic (CD8+) and regulatory T cells (FOXP3+). Percentages of CD8+ and FOXP3+ cells were quantified with QuPath, and infiltration patterns were assessed by pathologists. Results were correlated with overall survival (OS) and response to treatment after biopsy, which was defined according to RECIST criteria. As expected, metastases had less CD8+ (p=0.02) and FOXP3+ (p=0.007) cells than primary tumors. While CD8+ cells strongly correlated between primary and metastatic tumors (r=0.71, p=0.03), increased FOXP3+ cells in metastases were associated with high CAF regions (p=0.02). CD8+ cells alone were not prognostic; however, infiltration into the epithelium of metastases significantly improved OS (p=0.02). There was a strong correlation between CD8+ and FOXP3+ cell proportions in metastases (r=0.68, p<0.0001) not seen in primary tumors. CD8:FOXP3+ cell ratios in metastases correlated with longer OS (r=0.51, p=0.006) and were increased in patients with stable disease (p=0.02). These findings support the hypothesis that TIL characteristics may significantly affect mTNBC outcomes. We demonstrate that, although metastases have reduced immune presence, a higher CD8:FOXP3+ T cells may indicate a better prognosis in the metastatic setting. Furthermore, the correlation between CD8+ and FOXP3+ cells in metastases across sites, not observed in primary tumors, suggests that immunogenicity in metastases may be governed by different mechanisms than those in primary tumors, possibly contributing to the therapy resistance and worse outcomes observed in metastatic setting. The increased presence of FOXP3+ cells in metastases with high CAF levels suggests that metastatic CAFs promote an immunosuppressive environment, highlighting them as potential therapeutic targets. By identifying CAF-derived factors associated with increased FOXP3+ cell presence, novel therapies may help to overcome immunosuppression, enhance the cytotoxic immune response, and improve treatment efficacy in mTNBC. Citation Format: Alexandra D. Goudreau, Alexandre Penoit, Jade Marie Lasiste, Olga Aleynikova, Khaled Katergi, Josiane Lafleur, Adriana Aguilar-Mahecha, Mark Basik. Primary and metastatic tumors from triple negative breast cancer possess distinct immunological profiles [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 B025.

Abstract C013: Single cell analysis reveals tumor size-driven immune changes in primary breast tumors and corresponding lymph nodes

Abstract Introduction A sentinel lymph node is the primary node draining the tumor and is likely affected early in the metastatic process. The sentinel node holds a key position in the immune response against tumor in breast and represents a unique connection between the tumor and the host immune response. Detection of lymph node metastasis is important for staging a patient’s disease and is closely associated with prognosis. Although the clinical significance is debated, isolated tumor cells (<0.2 mm) appear to have no impact on survival while macro-metastasis (>2 mm) are linked to a poorer prognosis. Alterations in the immune response in lymph nodes with large versus small metastases have been observed, but little is known about the relationship between the immune response in the primary tumor and the metastatic status of the corresponding lymph nodes. Methods We analyzed single-cell suspensions from primary tumors and lymph nodes of 38 patients with early, operable breast cancer enrolled in the OSLO2 clinical observational trial using mass cytometry with a 47- antibody immune panel (including PanKeratin and EpCAM). In total 3.7 million single cells were analyzed and clustered into 40 metaclusters using FlowSOM. A second set of 22 metastatic lymph nodes from the OSLO2 cohort was used to validate results form the metastatic tumor cells. Additionally, immunofluorescence with E-cadherin and PanKeratin was applied, and intensity measured in 4 metastatic lymph nodes and imaging mass-cytometry was applied on one metastatic lymph node Results We found that the immune microenvironment in primary tumor was dominated by CD4 and CD8 T cells, particularly of exhausted and memory phenotype and with a higher frequency of activated regulatory T cells (Treg). In contrast, the immune composition in lymph nodes was predominantly composed of naturally occurring naive B and T cells. Notably, in line with previous publications we observed a skewing towards memory and exhausted phenotype in T cells, along with increased abundance of activated Treg in lymph nodes with larger metastasis to those with smaller or no metastasis. Furthermore, there were no correlation between the immune profile in the primary tumor and corresponding lymph node. The immune profile in the primary tumor was influenced by tumor size, with larger tumors showing a higher frequency of activated Tregs and exhausted CD8 T cells. Furthermore, In situ analysis revealed that the microenvironment in smaller metastase had an mesenchymal phenotype, while larger tumor deposits exhibited a more epithelial-like phenotype. ConclusionThe immune profile of the primary tumor did not predict the immune profile or metastatic status of the corresponding lymph node. Tumor size in both primary tumors and metastatic lymph nodes is the main drivers of changes in immune cell composition. Tumor cells from smaller metastases exhibited an mesenchymal phenotype, while larger metastasis displayed an epithelial phenotype. Citation Format: Inga Hansine H. Rye, Marit Otterlei Fjørtoft, Inger Riise Bergheim, Karin Teien Lande, Kanutte H. Huse, June Helen H. Myklebust, Ole Christian H. Lingjærde, Øystein. Garred, Jon H. Lømo, Hege H. Russnes. Single cell analysis reveals tumor size-driven immune changes in primary breast tumors and corresponding lymph nodes [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 C013.

Abstract A004: E2F5 conditional knockout in mammary epithelium drives organotropic metastasis in breast cancer

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 A006: Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer death in the United States with a 5-year survival rate of only 13%. Early diagnosis is very difficult and thus 53% of patients are diagnosed after metastasis has already occurred, with the liver being the most frequently affected site. Both primary tumors and metastases have dense desmoplastic stroma due to a high percentage of fibroblasts within the tumor microenvironment (TME). Previously, we developed a tunable 3D biomimetic model of the liver metastatic niche (LMN) to investigate how local fibroblasts affect tumor growth and chemoresistance in PDAC liver metastasis. This study aims to investigate the contributions of fibroblasts at the primary versus metastatic sites. We used matched primary PDAC and liver metastatic (LM) organoids derived from the Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre (“KPC”) genetically engineered mouse model. This allows us to compare the effects of primary PDAC cancer associated fibroblasts (CAFs) and liver metastasis associated fibroblasts (MAFs) on tumor cell growth and chemoresistance. Rheometry analyses were conducted to study the impact of local fibroblasts on the extracellular matrix (ECM) stiffness. Our results showed that both storage and loss moduli were enhanced only when PDAC CAFs were present in the co-cultured gels compared to gels with MAFs. Additionally, the 3D biomimetic model showed fibroblasts increased tumor growth and chemoresistance to gemcitabine, only when they were derived from the same site as the tumor cells. This highlighted the importance of studying the organ-specific microenvironment to develop better treatment options for patients with metastatic PDAC. To delve deeper into the molecular basis of these observations, we conducted RNA-sequencing to identify and compare the gene expression profiles in primary and metastatic tumor organoids exposed to conditioned media from CAFs and MAFs using a transwell membrane co-culture model. Our results showed significant differences in gene expression based on the type of fibroblasts co-cultured with the organoids. Moreover, we investigated the role of MAF-derived exosomes on LM growth and chemoresistance using exosome depletion and direct exosome addition to LM organoids. Lastly, we performed exosomes size and distribution characterizations using dynamic light scattering (DLS) analysis. Taken together, these findings highlight the critical role of the organ-specific microenvironment in tumor progression and the necessity of targeted therapies to improve patient outcomes in metastatic PDAC. Our results underscore the importance of developing a tunable 3D biomimetic model of the LMN, as it enables the testing of novel therapeutic strategies designed to disrupt interactions between MAFs and tumor cells, and potentially advancing treatments for metastatic PDAC. Citation Format: Mahsa Pahlavanneshan, Weikun Xiao, Eileen Fung, Vaidhyanathan Mahaganapathy, Chae Young Eun, Chang-Il Hwang, Shannon Mumenthaler, Reginald Hill. Investigating the effects of fibroblasts derived from the primary versus the metastatic organ on 3D biomimetic models of pancreatic 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 A006.

Abstract C001: Cytomegalovirus infection modulates distinct immune cell dynamics across tumor-immune microenvironments and systemic macroenvironment during metastasis

Abstract Cytomegalovirus (CMV) is ubiquitously present worldwide and has been implicated in the modulation of cancer development and spread. Notably, CMV proteins have been detected in approximately 90% of lymph node and brain metastases in breast cancer, suggesting its potential influencing tumor invasiveness and metastases. This study investigates the effects of CMV infection on the tumor microenvironment and macroenvironment during metastatic progression in triple-negative breast cancer (TNBC). We employed a syngeneic TNBC mouse model designed to develop spontaneous metastases following primary tumor resection. Using multi-omics sequencing and comprehensive analysis tools, we characterized the tumor-immune landscape across multiple organs relevant to CMV infection and tumor metastasis, including blood, bone marrow, spleen, lungs, brain, and lymph nodes. Our results demonstrate that CMV infection systematically activated the immune response, suppressing primary tumor growth and subsequent metastasis. At the primary tumor site, prior to metastasis, CMV-infected mice exhibited significantly lower proportions of macrophages and plasma cells compared to uninfected counterparts, while CD8+ T cells were significantly enriched. In contrast, the proportions of neutrophils and dendritic cells did not significantly differ between the groups. Distinct patterns of immune cell dynamics were observed at metastatic sites. In the spleen of CMV-infected mice, a notable reduction in macrophages and elevated neutrophil levels were detected. Lung tissues displayed a marked increase in macrophages, CD8+ T cells, and dendritic cells in CMV-infected mice. Interestingly, in the blood, we noted a decreased proportion of natural killer cells in CMV-infected mice. However, no significant difference in plasma cell percentages was observed between CMV-infected and uninfected mice in metastatic lung tissues. The cell-cell interaction analysis further showed a complex crosstalk network between immune cells as well as tumor and immune cells. These findings highlight the profound impact of CMV infection on remodeling the immune landscape at both primary tumors and metastatic sites. The distinct immune cell dynamics orchestrated by CMV infection underscore the virus's role in modifying tumor-immune microenvironments and macroenvironments. Our study offers novel perspectives for developing targeted cancer therapies that consider the complex interactions between viral infections and the tumor immune axis. Further research is warranted to elucidate the mechanisms underlying CMV-mediated immune modulation at molecular level and its potential therapeutic implications in TNBC and other cancer types. Citation Format: Wenjuan Dong, Jianting Sheng, Shan Xu, Matthew Vasquez, Hong Zhao, Stephen T.C Wong. Cytomegalovirus infection modulates distinct immune cell dynamics across tumor-immune microenvironments and systemic macroenvironment during 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 C001.

Abstract PR001: An immunomodulatory crosstalk between cancer cells and the hepatic microenvironment underlies mutant estrogen receptor-driven breast cancer-to-liver metastasis

Abstract While major advancements have been made in treatment of primary tumors in estrogen receptor-positive (ER+) breast cancer, reducing mortality from metastatic breast cancer (mBC) in treated patients who develop endocrine resistance to first line therapies remains an unmet clinical need. Wild-type ER activity or availability of its cognate ligand 17-beta estradiol (E2) are major factors in primary tumor progression, which are lost/reduced due to endocrine treatments, such as use of selective estrogen degraders (SERDs) and aromatase inhibitors (AIs), respectively. Over time, this leads to selection of rare cancer cells bearing point mutations in ESR1, the gene encoding ER-alpha, which become the dominant population at metastatic sites, and such mutations are now recognized as a frequent driver of endocrine resistance and metastasis, especially in patients with advanced ER+ breast cancer who have been heavily pre-treated with AIs. Analyses of clinical samples have shown a statistically significant association enrichment of ESR1 mutations in liver metastases, compared to the primary tumor. Other distant tissues commonly colonized by breast cancer cells, such as bones, lungs, or brain, although prevalent, do not display this strong association. While this might be due to ease of biopsy sampling in the liver compared to other sites, an alternate and intriguing hypothesis is that the liver offers a fertile soil for metastatic colonization by mutant ER-expressing cells, compared to other distant sites, such as lungs. Using a combination of pre-clinical models, we assessed if mutant ER could drive metastatic colonization at distant sites, such as the liver. We have uncovered a novel link between cancer cell-intrinsic immunomodulatory signaling pathways that are specifically upregulated in mutant ER-expressing breast cancer cells, which represents at least one major molecular mechanism underlying the observed liver tropism of mutant ER-associated, endocrine resistant ER+ breast cancer. We show that disseminated, mutant ER-expressing BC cells engage in crosstalk with the liver stromal and immune populations post-extravasation to further fuel metastatic outgrowth. Citation Format: Sunny Das, Joana Liu Donaher, Lisa Verhoeven, Ranjan Mishra, Ferenc Reinhardt, Sumeet Gupta, Zheqi Li, Kornelia Polyak, Robert A. Weinberg. An immunomodulatory crosstalk between cancer cells and the hepatic microenvironment underlies mutant estrogen receptor-driven breast cancer-to-liver 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 PR001.

Abstract B045: Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem cells

Abstract Background: Breast cancer is the most frequently diagnosed cancer, and the second leading cause of cancer-related death in women. Most breast cancer related deaths are due to metastasis to distant organs. The primary tumor and secondary site microenvironments provide cues that activate cancer cell plasticity programs such as those associated with invasion and stemness, which are needed for dissemination and the establishment of metastatic foci. We demonstrated that in the primary tumor >50% of cancer stem cells (CSCs) co-express the invasive phenotype marker, MenaINV. Given that ∼80% of newly arriving circulating tumor cells to the lung are CSCs, we questioned if extravasation of CSCs to the lung is mediated solely by MenaINV or if other compensatory pathways are at play. We describe here our discovery that CSCs require MenaINV for extravasation to the lung parenchyma. Methods: To visualize breast cancer stem cell extravasation into the lung parenchyma, we expressed the stemness biosensor, SORE6, in MDA-MB-231 breast cancer cells. SORE6 is composed of 6 tandem repeats of the Sox2 and Oct4 response elements placed upstream of destabilized Green Fluorescent Protein (GFP). To distinguish CSCs (green) from non-CSCs, all cells were also labeled with red tracker dye. Breast cancer cells transduced with lenti-SORE6 were FACS sorted into low SORE6-GFP cells (non- CSCs) and high SORE6-GFP (CSCs). Activation of the stem program in FACS sorted cells was validated by qRT-PCR analysis of the widely used stem cell markers CD44, CD133, P63, ALDH1, Sox9, Sox2, and Oct4. The extravasation efficiency of non-CSCs vs. CSCs was assessed in vitro by the extravasation directed transendothelial migration (eTEM) assay, which mimics extravasation by allowing migration of cancer cells through endothelial cells cultured on a porous membrane, as well as in vivo, by intravital imaging. The impact of MenaINV on CSC extravasation was evaluated through CRISPR/Cas9 mediated knockout of MenaINV. In in vivo experiments, a 50/50 mixture of non-CSCs and CSCs expressing either wild type or knocked-out MenaINV were injected into the vasculature of Rag2-/- MacBlue mice. in vivo visualization of cancer cell extravasation was accomplished by implantation of an indwelling lung imaging window prior to the injection of cancer cells, followed by high-resolution intravital imaging. Results: CSCs exhibit significantly higher expression of all stem cell markers and express more MenaINV compared to non-CSCs. Moreover, CSCs show significantly better transendothelial migration in eTME assay (p=0.03) and lung extravasation (p<0.0001) in intravital imaging, compared to non-CSCs. Finally, knocking out MenaINV reduced lung extravasation to the levels comparable to non-CSCs. Conclusions: This study highlights the crucial role of MenaINV in facilitating lung metastasis by CSCs. Thus, targeting MenaINV may represent a promising therapeutic strategy to reduce metastatic burden in breast cancer patients. Citation Format: Mohd Nauman, Yookyung Jung, Burcu Karadal-Ferrena, Camille Duran, Madeline Friedman-DeLuca, Nicole Barth, Prachiben Patel, John Condeelis, David Entenberg, Maja H. Oktay. Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem 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 B045.

Effects on lymph node size, staging and primary tumor histology on diagnostic accuracy of axillary lymph node aspirate of breast cancers.

Fine-needle aspiration cytology is preferred for axillary lymph node metastasis with low costs and minimal risks. To improve diagnostic performance by incorporating clinical-radiological-pathological parameters, a large cohort pre-operative aspirates in were reviewed for parameters affecting adequacy rate and accuracy. Axillary nodal aspirates from three institutions with histologic correlation were retrieved. Case notes were reviewed for parameters pertaining to the primary tumor, nodal status, histologic and cytologic diagnoses. Totally 1361 specimens were included. The risk of malignancy for C1-C5 categories were 53.39%, 27.45%, 70.97%, 83.33% and 88.00%, increasing to 75.86%, 94.59% and 99.28% for C3/C4/C5 categories excluding cases with neoadjuvant therapy. Node size (p < 0.001) and histologic grade (p = 0.003) of primary tumor positively correlated with specimen adequacy. Presence of in situ component trended towards inadequacy (p = 0.069). Lymph node size remained a strong predictor of concordant cytologic diagnosis (p < 0.001). A higher percentage of involved node (p = 0.006) and HER2 overexpressed breast cancers (p = 0.027) increased concordance. Cases with ≥ 4 (up to ≥ 10) positive nodes were more likely to be concordant (p = 0.009- < 0.001), with improvements of 8.27%-12.37%. For size, cut-offs of ≥ 5 and ≥ 10mm were significant (p = 0.006- < 0.001). It is critical that clinical-radiological-pathological findings be interpreted together with cytology. Aspirates from smaller nodes are more likely to be non-informative, irrespective of the total number of suspicious nodes, or a high-grade primary. In axillae with less than 4 suspicious nodes and/or a target node of less than 5-10mm, the diagnostic accuracy of aspiration cytology decreases and should be interpreted cautiously.

Abstract C037: Chemotherapy causes de novo induction of invasive breast cancer cells

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 C018: Awakened dormant cancer cells undergo highly mesenchymal to quasi- mesenchymal transition and acquire stemness

Abstract The awakening of dormant disseminated cancer cells is likely responsible for the clinical relapses of patients whose primary tumors have been cured months and even years earlier. In the present study, we demonstrate that dormant breast cancer cells lodged in the lungs reside in a highly mesenchymal, non-proliferative phenotypic state. The awakening of these cells does not occur by a cancer cell-autonomous process. Instead, inflammation and wound healing of the surrounding tissue microenvironment causes them to shift from a highly mesenchymal to a quasi-mesenchymal phenotypic state in which they acquire stemness and proliferative ability. Once awakened, these cells can stably reside in this quasi-mesenchymal state and maintain their stemness, doing so without ongoing heterotypic signaling from the lung microenvironment. EGFR ligands released by the cells of the injured tissue microenvironment, including notably M2 type macrophages, promote dormant cancer cells to move toward this quasi-mesenchymal state, a transition that is essential for the awakening process. An understanding of the mechanisms of metastatic awakening may lead in the future to treatment strategies designed to prevent such awakening and resulting metastatic relapse. Citation Format: Jingwei Zhang, Robert Weinberg. Awakened dormant cancer cells undergo highly mesenchymal to quasi- mesenchymal transition and acquire stemness [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 C018.

Abstract B023: Regulatory T (Treg) cell contributes to tumor cell dissemination via extracellular matrix (ECM) remodeling, which is driven by TAMs in an IFNg-dependent manner

Abstract Background: The extracellular matrix (ECM) is a highly organized non-cellular network consisting of structural proteins, growth factors, cytokines and other secreted molecules. The ECM is highly dynamic and is a critical player in the regulation of local invasion and metastasis. The ECM network is ever-changing and an important immunosuppressive component of the tumor microenvironment (TME). However, the reciprocal effects of the immune system on the tumor-associated ECM are elusive and largely unexplored. Regulatory T (Treg) cells function to enforce peripheral tolerance and are potent suppressors of tumor immunity. We have previously shown that Treg cells promote tumor growth in murine breast cancer models by favoring alternative activation of tumor-associated macrophages (TAMs) via suppression of IFN-g. Aims: In this work, we sought to assess whether Treg cell-mediated immune suppression is associated with changes in the ECM, and whether those ECM changes functionally impact metastatic dissemination. Further, we assessed the requirement of TAMs and IFN-g in the ECM remodeling. Design Methods: In this study, we used spontaneous and transplantable models of breast cancer susceptible to genetic ablation of Treg cells. We used a combination of histopathological analysis, tumor decellularization, bioengineering 3D-chip modeling, in vivo manipulations and bioinformatic analysis, to evaluate remodeling of the ECM, cancer cell invasive behavior and metastatic dissemination. Results Summary: Treg cell ablation resulted in significant ECM remodeling, with reduced amounts and organization of collagen fibers, and increased amounts of fibronectin and laminin. Tumor cells seeding on Treg cell ablated tumor- derived decellularized ECM matrices results in reduced epithelial-mesenchymal transition (EMT) transcription factors, and profound impairment of collective migration. In vivo, Treg cell ablation in a neo-adjuvant setting followed by primary tumor resection resulted in significant reduction of circulating tumor cells (CTC) and impairment of lung metastatic disease. Importantly, we demonstrated that Treg cell ablation-driven changes in the matrisome correlate with long-term improved survival in a cohort of breast cancer patient samples. Through genetic knock-out models and in-vivo neutralization, we observed that Treg cell-dependent changes in ECM-related phenotypes are dependent on IFN-g-signaling. Single-cell RNA-sequencing (scRNA-seq) of murine breast tumors revealed that the matrisome signature is highly upregulated among TAMs, compared to other TME cell types. Lastly, using conditional genetic knock-out models we show these IFN-g-dependent changes are mostly driven by TAMs. Conclusions: Altogether, we identified a novel metastasis-promoting effect of Treg cells in the breast cancer microenvironment through regulation of ECM dynamics beyond their described effects on primary tumor growth. Citation Format: Ailen D. Garcia-Santillan, Jessanne Y. Lichtenberg, He Shen, Jasmine M Rodriguez, Jonathan Barra, Nicholas M Clark, Wei Du, Leandro M Martinez, Ashley D Hadjis, Taylor Calicchia, Mikhail G Dozmorov, Jose J Bravo-Cordero, Amy L Olex, Priscilla Y Hwang, Paula D Bos. Regulatory T (Treg) cell contributes to tumor cell dissemination via extracellular matrix (ECM) remodeling, which is driven by TAMs in an IFNg-dependent manner [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 B023.

Abstract B040: Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer

Abstract Breast cancer is one of the most frequently diagnosed cancers and the second leading cause of women’s cancer death. Although the five-year survival rate for women in the United States with nonmetastatic invasive breast cancer is 90%, this rate drops to less than 30% for those with tumors that have metastasized to distant organs. Epithelial-to-Mesenchymal Transition (EMT) plays a critical role in tumor metastasis by enabling the loss of epithelial characteristics and the gain of mesenchymal traits, leading to increased cell motility, invasion, and resistance to therapy. Consequently, targeting the EMT process may prevent tumor cell migration and improve patient outcomes. Rather than being a binary switch from an epithelial to a mesenchymal state, cells undergoing EMT can assume a spectrum of intermediate states, some of which may reshape the tumor microenvironment by recruiting pro-tumor immunosuppressive cells, such as regulatory T cells, M2-like macrophages, and myeloid-derived suppressor cells. However, the impact of EMT on the immune composition of the tumor microenvironment and the causative mechanisms remain poorly understood. Using multi-omics approaches in a mouse model of triple-negative breast cancer, we assessed the interaction between tumor and immune cells in the tumor microenvironment driven by the EMT. A heterogeneous mouse breast cancer cell line (4T1) was used to isolate five distinct single-cell-derived clonal populations residing in distinct states within the EMT spectrum. These EMT clones, along with the parental 4T1 cell line, were orthotopically transplanted into the mammary fat pads of BALB/c mice. Primary tumors were subsequently harvested and dissociated for single-cell RNA-sequencing (scRNA-seq) and DNA methylation analysis. Lungs were histologically evaluated for metastases. Tumors derived from an intermediate EMT clone exhibited increased tumor growth kinetics and a higher incidence of metastasis compared to epithelial or mesenchymal clones. In scRNA-seq data, tumors derived from different EMT clones largely retained their original EMT states in vivo and recruited distinct immune cell profiles. DNA methylation analysis further revealed that these tumors displayed unique methylation patterns in EMT-related CpGs. This study introduced a model that captures the complexity of the tumor microenvironment across the EMT spectrum. This was accomplished through immunophenotyping using scRNA-seq and identifying unique epigenetic patterns through DNA methylation. Our findings highlight the potential for distinct immune cell populations to be recruited to tumors in different EMT states, thereby influencing survival and metastasis outcomes. Furthermore, our results open new opportunities for developing cancer combination treatment strategies that target specific tumor-immune cell interactions. Citation Format: Hanxu Lu, Meisam Bagheri, Todd Miller, Diwakar Pattabiraman, Brock Christensen. Investigating the impact of tumor EMT states on immune cell infiltration 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 B040.

Abstract IA016: Vascular control of metastasis

Abstract The molecular analysis of tumor vessel interactions during tumor progression and metastasis has primarily focused on the study of tumor cell-derived angiogenic and lymphangiogenic signals with the purpose to exploit such factors as therapeutic targets. Angiogenic factors activate endothelial cells in nearby blood and lymphatic capillaries to sprout towards the tumor. Tumor angiogenesis thereby not only nourishes the growing tumor, but access to the blood and lymphatic vasculatures enables cells from the primary tumor to enter the circulation to eventually form metastases at distant sites. The complex cellular interactions between tumor cells and endothelial cells have in this context mostly been studied from a tumor cell-centric perspective, i.e., the tumor cells send signals to which endothelial cells merely respond. Yet, the past decade has witnessed a fundamental change of paradigm with the discovery that the vascular endothelium does not just respond to exogenous cytokines but exerts active ’angiocrine’ gatekeeper roles controlling their microenvironment in an instructive manner. We have applied the concepts of angiocrine signaling towards the study of tumor progression and metastasis. Employing novel surgical preclinical metastasis models that better mimic tumor progression and the response to therapy as it occurs in humans, we have molecularly dissected vascular endothelial cells in progressing primary tumors as well as in the pre-metastatic and metastatic niches. These experiments were on the one hand aimed at establishing the systems map of endothelial transcriptomic changes during tumor progression and metastasis and on the other hand to identify and validate novel therapeutic targets. This presentation will review recent advances in the field of tumor microenvironment research and present novel angiocrine signaling mechanisms as promising targets of future mechanism-driven anti-metastatic therapy. Citation Format: Hellmut G. Augustin. Vascular control of 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 IA016.

Abstract C032: The impact of neoadjuvant endocrine therapy (NET) in ER+ breast cancer tumor microenvironment and metastasis

Abstract Breast cancer is the most common cancer in women worldwide and the second leading cause of death. The Estrogen Receptor positive (ER+) subtype constitutes approximately 80% of the yearly 250,000 breast cancer diagnoses in the US. Anti-estrogen endocrine therapy, administered either pre- or post-operatively, is the cornerstone treatment for all stages of breast cancer. Pre- operative or neoadjuvant endocrine therapy (NET) reduces pre-surgical tumor burden, improves surgical outcome, and provides prognostic information. Although the treatment often has a favorable initial response, about 20% of patients eventually develop resistance and recurrent disease, even decades after the primary diagnosis.We hypothesized that resistance in NET is mediated through pro-metastatic changes in the tumor microenvironment affecting either (i) dissemination machinery or (ii) cancer cell phenotype.The main mode of breast cancer hematogenous dissemination to distant sites is through intravasation portals called Tumor Microenvironment of Metastasis (TMEM) doorways. Each TMEM doorway consists of a tumor cell overexpressing the actin-regulatory protein MENA, a Tie2high/ VEGFAhigh macrophage and an endothelial cell, all in direct contact. TMEM doorway activity leads to vascular opening events, during which tumor cells located in areas around TMEM doorways intravasate and disseminate to secondary sites. Therefore, to investigate the impact of NET on cancer cell dissemination, we evaluated its effect on (i) TMEM doorway score (the number of TMEM doorways per tissue area), (ii) TMEM doorway opening, (iii) the number of circulating tumor cells and (iv) the number of disseminated tumor cells. We used the orthotopic MCF7 breast cancer xenograft model grown in NOD/SCID mice and tested two drugs widely used in NET, Fulvestrant (a Selective Estrogen Receptor Degrader) and Tamoxifen (a Selective Estrogen Receptor Modulator). NET did not affect the TMEM doorway score, TMEM doorway activity, the number of CTCs or the number of DTCs. We are now focused on evaluating the effect of NET on the density of cancer cells which are upregulated in programs of invasion, stemness, and dormancy. This “Triple Threat Phenotype (TTP)” gives tumor cells an enhanced ability to disseminate to and colonize distant organs. To this effect we will stain primary MCF7 tumors from NET and vehicle-treated mice for markers of invasiveness (MENA), stemness (SOX9) and dormancy (NR2F1). Detecting any changes in the phenotype of metastatic tumor cells following NET will help us understand underlying mechanisms of endocrine therapy resistance and may lead to new therapeutic targets and improve patient outcomes. Citation Format: Anastasia Aristoteleia Chondronikola, Dimitra Anastasiadou, Aliona Zintiridou, Camille L. Duran, Nicole Barth, Burcu Karadal-Ferrena, Erika Pereira-Zambalde, Lina Ariyan, Suryansh Shukla, George S. Karagiannis, David Entenberg, John S. Condeelis, Maja H. Oktay. The impact of neoadjuvant endocrine therapy (NET) in ER+ breast cancer tumor microenvironment and 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 C032.

Abstract C034: Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors

Abstract Although various effective anti-cancer treatments have become available over the last decades, resistance to all available therapies remains the major cause of death of cancer patients with disseminated tumors. Striking examples are patients with triple-negative breast cancer (TNBC), which are frequently defective in the repair of DNA double strand breaks, e.g. due to loss of BRCA1 function. Because of this defect, the patients initially respond very well to DNA damage-inducing chemotherapy. Unfortunately, disseminated tumors are usually not eradicated and resistant tumor cells are eventually selected from residual primary or metastatic tumor sites. It is therefore crucial to understand the molecular mechanisms underlying the drug tolerance of the residual tumor cells. To study residual disease, we used the K14cre;Brca1 F/F ;p53 F/F (KB1P) mouse model for hereditary breast cancer, which provides the unique opportunity to explore and target those mechanisms in a fully immunocompetent model. The mammary tumors that spontaneously develop highly resemble their human counterparts, both morphologically and in their therapy response. For example, tumors shrink in response to poly(ADP-ribose) polymerase inhibition, platinum-based treatment or the commonly used doxorubicin, docetaxel and cyclophosphamide (TAC) combination therapy. But despite repeated drug sensitivity, the KB1P mammary tumors are not eradicated, not even by a frequent dosing schedule. By combining single-cell RNA sequencing, spatial transcriptomics and imaging mass cytometry, we dissected the intratumoral heterogeneity and alterations in the tumor microenvironment of residual disease. We identified specific subpopulations of tumor cells that have a survival benefit after treatment, and these occurred together with an altered microenvironment characterized by a highly reactive stroma infiltrated with both anti-inflammatory and pro-tumoral immune cells. Interestingly, those structural and transcriptional changes are reversed in the relapsed tumors, highlighting the plasticity of drug tolerance. To investigate the mechanisms of the altered tumor-stroma interactions that are relevant for drug tolerance, we have designed a custom-made CRISPR/Cas9 library based on differential gene expression of the residual tumor cells. This library enables us to functionally test relevant mechanisms of drug tolerance in vivo and we expect that these analyses will provide useful insights into the tumor-stroma interactions that contribute to residual disease. Taken together, our detailed analyses demonstrate the substantial remodeling of tumor cells in their microenvironment upon treatment. To develop new therapeutic approaches to eradicate drug-tolerant tumor cells and thereby circumvent tumor relapse, it is essential to better understand the heterogeneous cellular composition of residual tumors in its spatial context. With this project, we hope to provide comprehensive data to develop better therapeutic strategies that target the tumor-stroma interaction and eradicate residual tumors. Citation Format: Morgane Decollogny, Demeter Túrós, Astrid Chanfon, Myriam Siffert, Ismar Klebic, Sven Rottenberg. Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors [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 C034.

Abstract C010: Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in head and neck squamous cell carcinoma

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&amp;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 C004: Glucocorticoids establish a metastatic-promoting tumor microenvironment in a PDAC mouse model

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 &amp;lt;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.