Stromal Tumors Research Articles

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 287.

Abstract 1393: Tumor-infiltrating monocyte-like cells create metastasis-promoting immunosuppressive microenvironment via THBS1 production in aggressive colorectal cancer

Abstract Mesenchymal subtype of colorectal cancer (CRC) is characterized by high metastatic potential, poor prognosis and an immunosuppressive tumor microenvironment (TME). Although tumor-infiltrating myeloid cells constitute the primary population of immune components in the TME, targeting myeloid cells remains highly challenging owing to the developmental and functional heterogeneity of their subsets. Thus, a deeper understanding of how tumor-infiltrating myeloid cells contribute to the creation of a tumor-promoting TME is needed to identify rational strategies against CRCs with aggressive phenotypes. Recent studies have revealed that multiple inflammatory signatures are elevated in mesenchymal CRCs, suggesting a strong association between inflammation and stromal activation. In this study, we focus on thrombospondin-1 (THBS1), which is a matricellular protein, typically expressed in inflammatory processes. Immunostaining and RNA in situ hybridization on human CRC samples revealed a clear localization of THBS1 expression in tumor stroma. Analyses of the cancer genome atlas (TCGA) and tissue microarray of human CRCs show a positive correlation between increased expression of THBS1 and the mesenchymal phenotype, immunosuppression, and poor prognosis in human CRCs. Utilizing immunocompetent orthotopic implantation systems with aggressive mouse tumor organoids (MTO) on wild-type and Thbs1 knockout (Thbs1−/−) mice, we demonstrated that loss of THBS1 in the TME suppressed metastatic formation accompanied by an increased infiltration of tumor-infiltrating CD8+ T cells. Depletion of CD8+ T cells by neutralizing antibody restored metastases in MTO-bearing Thbs1−/− mice, indicating that THBS1 promotes metastatic formation by suppressing CD8+ T cell-mediated anti-tumor immunity. Analyses of single cell RNA sequencing data of human CRCs and orthotopic MTO tumors revealed that bone marrow (BM)-derived monocyte-like cells are the primary source of THBS1. This notion was further supported by analyzing the orthotopic MTO tumors in chimeric mice generated by bone marrow transplantation. Finally, we identified stroma-derived CXCL12 as a key chemokine for the recruitment of THBS1-producing monocyte-like cells by analyzing orthotopic MTO tumors treated with CXCL12 inhibitor. Our study contributes to a better understanding of the processes underlying THBS1 production by monocyte-like cells that mediates immune evasion that confer aggressive features to CRCs. These findings provide insights for the treatment of this poor-prognosis malignancy. Citation Format: Mayuki Omatsu, Yuki Nakanishi, Kosuke Iwane, Naoki Aoyama, Takahisa Maruno, Hiroaki Kasashima, Masakazu Yashiro, Akihisa Fukuda, Hiroshi Seno. Tumor-infiltrating monocyte-like cells create metastasis-promoting immunosuppressive microenvironment via THBS1 production in aggressive colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1393.

Abstract 4212: Mimicking HCC complex biology and diverse treatment response in a patient-derived microfluidic model

Abstract Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is closely associated with progressive stages of liver diseases. Its transformation, survival, progression and metastasis have been associated with regulation mechanisms orchestrated by the tumor stroma. Patient differences in tumor composition and cellular signaling are reflected in the diversity of therapy response. Hence, comprehensive in vitro models that capture patient-specific tumor and stromal components are necessary to elucidate and evaluate anticancer therapy. Patient-derived dissociated HCC cells from eight different patients were combined with tumor-derived fibroblasts and endothelial cells within a microfluidic setup comprising 64 parallel chips in a microtiter plate format. Cultures were challenged with an individual or combinatorial treatment from a panel of tool compounds for 72 hours in an automated setup. We used immunofluorescence and high content confocal imaging to assess the structural and biological interaction between cancer cells and the associated stroma. Viability assessment, artificial intelligence-based vascular morphology characterization and multiplexed chemokine/cytokine release measurements were used to elucidate patient and drug-dependent effects of single or combinatorial treatments. Cellular interaction within the microfluidic platform led to a vascularized tumor construct, with hepatocellular carcinoma aggregates being enveloped and traversed by a lumenized and interconnected vascular plexus, in association with tumor-derived fibroblasts. Compound treatment led to profound differences across the tested parameters, indicating different anticancer effects in conjunction with the biological diversity of the patient material. Finally, morphological assessment revealed distinct compound-induced effects on the tumor’s vascular organization. We present a vascularized patient-derived HCC model that includes relevant cellular players of the tumor microenvironment found in vivo. These co-cultures are highly suitable for studying specific cell types as well as patient-specific responses. We envision that this system has the potential to provide a platform for understanding the interplay between different cell types present in hepatocellular carcinomas, with sufficient scalability ease of use for industrial and clinical implementation. Citation Format: Orsola Mocellin, Abbie Robinson, Aleksandra Olczyk, Stephane Treillard, Thomas Olivier, Chee P. Ng, Jeroen Heijmans, Arthur Stok, Gilles van Tienderen, Monique Verstegen, Sebastian J. Trietsch, Henriëtte L. Lanz, Paul Vulto, Jos Joore, Karla Queiroz. Mimicking HCC complex biology and diverse treatment response in a patient-derived microfluidic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4212.

Abstract 5150: An immunohistochemical survey of predictive biomarkers for immunotherapy in ampullary carcinoma identifies strata with unique prognostic signatures

Abstract Introduction: Carcinomas located in the ampulla of Vater are a low incidence malignancy. Therapeutic options for early stage disease largely consist of pancreaticoduodenectomy followed by adjuvant chemotherapy with or without radiation. We explored the localization of CD8 and CD3 positive cells in addition to PDL-1 staining and mismatch repair deficiency markers to gain insight into the potential applicability of immunotherapy for patients with this disease. Methods: Excess material from clinically diagnosed cases collected between 1997 - 2013 were used to construct a duplicate 0.6mm core tissue microarray (TMA). All cases were derived from the Vancouver Coastal Health Region. Immunohistochemical staining was performed with the following markers: CD8, CD3, PD-L1, and the mismatch repair (MMR) markers: MSH2, MSH6, MLH1 and PMS2. CD3 and CD8 were quantified using localization within the tissue. PD-L1 was considered positive with >1% of cells stating positive. MMR markers were confirmed using full section and cases with convincing loss of any MMR marker were considered MMR deficient. Univariable disease specific survival analysis was performed to identify unique prognostic signatures and contingency analysis was performed to identify co-expression of the aforementioned biomarkers. Results: Ninety-eight cases were evaluable for all markers. The median age of the cohort was 69 [41 - 84] years. Forty-two percent were female. Adjuvant chemotherapy was provided to 15% of patients with the remainder undergoing post-surgical observation. Almost 90% were pT3 or pT4 and 48% had regional lymph node metastasis. CD3+ T-cells were either localized in the tumor stroma only (59%) or in both the stroma and epithelial compartments (41%). CD8+ T-cells were principally located in the stroma (66%) and both the epithelial and stroma (24%). Ten percent of cases had no CD8+ T-cells. PD-L1 staining was found in 10% of cases. MMR deficiency was found in 5% of cases. Improved prognostic signatures were found for cases with CD3+ and CD8+ T-cells in both the epithelial and stromal compartments (p <= 0.02). A trend towards a negative prognostic signature was observed for MMR deficient cases and for PD-L1 positive cases. The only significant positive association between biomarkers was found for CD3+ and CD8+ in both the epithelial and stromal compartments. Conclusions: The results of this analysis suggest that enhancement of an immune response yielding an increase CD3+ and CD8+ T-cells in the epithelial compartment may have a beneficial effect on prognosis. The relative scarcity of MMR deficiency in this disease is within the expected prevalence supported by other studies. Further exploration of the potential role of immunotherapy in immune enhanced subgroups of ampullary cancers is warranted. Citation Format: Steve E. Kalloger, Joanna Karasinska, James Topham, Daniel J. Renouf, David F. Schaeffer. An immunohistochemical survey of predictive biomarkers for immunotherapy in ampullary carcinoma identifies strata with unique prognostic signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5150.

Abstract 7417: SpatialCS: A tool to effectively integrate histological annotations and spatial transcriptomics analysis

Abstract Spatial transcriptomics is revolutionizing our understanding of tumor ecosystems, providing insights into the spatial dimensions of cell locations, their organization, and interactions within tissues. A key challenge in this field is integrating histological annotations with spatial domains identified through unsupervised clustering methods like SpaGCN. This integration is crucial for accurate spatial domain identification and further analyses. However, clustering results often vary based on parameters such as resolution, leading to inconsistencies with histological annotations. This variation necessitates optimized parameter selection to achieve the best clustering outcomes. We here introduce SpatialCS, a novel tool specifically designed to both visualize and quantify the alignment between histological annotations and the clustering results obtained from spatial transcriptomics data. SpatialCS employs a new index based on the asymmetric Wallace indices. This index penalizes spot pairs that are clustered together but do not share the same histology annotation type, with penalties scaled according to their transcriptional similarity. Additionally, we propose a purity index, derived by calculating the average transcriptional entropy of all clusters. This index serves as a measure of the homogeneity within each cluster. Furthermore, SpatialCS includes a unique visualization function. This function projects histological annotations onto the clustering results as outlines, making it easier to assess the consistency between histology annotations and clustering outcomes. We have applied SpatialCS to Visium spatial transcriptomics data generated on four colorectal cancer liver metastases, each accompanied by comprehensive spot-level histological annotations in both tumor and normal compartments. The histologic annotations include but not limited to are tumor cells, tumor cell cluster subtypes, tumor stroma, tumor in the vessels, portal tract, individual bile ducts, portal vein and liver cell plates and non-neoplastic stroma. The application of SpatialCS has proven its effectiveness in identifying the most consistent clustering results, which were further confirmed by experienced pathologists. Notably, the output of SpatialCS offers helpful guidance for improving histological annotations. Together, SpatialCS not only provides a quantitative approach to evaluate the consistency between histological annotations and unsupervised clustering analysis but also showcases its potential to significantly enhance histological annotations. Citation Format: Yanshuo Chu, Kyung Serk Cho, Isha Khanduri, Tieling Zhou, Jiahui Jiang, Yunhe Liu, Xinmiao Yan, Guangsheng Pei, Yibo Dai, Yang Liu, Ruiping Wang, Akshaya S Jadhav, Sharia Hernandez, Humam Kadara, Luisa Maren Solis Soto, Scott Kopetz, Dipen Maheshbhai Maru, Linghua Wang. SpatialCS: A tool to effectively integrate histological annotations and spatial transcriptomics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7417.

Abstract 715: Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment

Abstract The stroma of solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell-derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Increasing evidence demonstrates their ability to hamper cancer patients' response to most treatments currently applied in the clinic, including immunotherapy. Therefore, strategies to counteract negative effects of TAMs are nowadays gaining momentum at preclinical, translational, and clinical levels. Here, we present the development and evaluation of nanoparticles (NPs) loaded with TLR agonists and/or inhibitors of immunosuppressive pathways to reprogram TAMs and the tumor microenvironment (TME) to unleash an effective immune response to fight against the tumor. Firstly, we have evaluated in vitro the ability of poly(I:C) and/or R848, agonists of TLR3 and TLR7/8 respectively, to reprogram TAMs into antitumor effector cells, and also the efficacy of Stattic and/or Galunisertib, inhibitors of STAT3 and TGF-β pathways respectively, to inhibit immunosuppression by cancer cells and/or TAMs. The best combinations of these drugs were encapsulated in nanoemulsions or polymeric nanocapsules for improved TAM-targeting and pharmacokinetics in vivo. These NPs were characterized for their physicochemical properties and also tested in vitro using primary human macrophages. For in vivo evaluation, subcutaneous and orthotopic murine models of lung cancer (CMT167) were used, showing antitumoral efficacy and TME reprogramming as evaluated by FACS, RNA analysis and multiplex immunofluorescence. Protamine-NCs-loaded with poly(I:C)+R848 and coated with an additional layer of hyaluronic acid functionalized with mannose were used to target the CD206 receptors, showing antitumoral efficacy mediated by TAM reprogramming, evaluated as higher CD86 while lower CD206 and Arg1 expression. Nanoemulsions with a PEGylated surfactant, encapsulating Stattic+Galunisertib+R848 showed faster antitumoral activity versus the free drugs. Experiments performed in IFN-γ KO mice and immune deficient mice (NSG and Balb/c nude) revealed that a fully functional immune system is crucial for the response to the treatment. In conclusion, our work demonstrates the antitumoral efficacy and reprogramming of the tumor microenvironment by combinations of TLR agonists and/or inhibitors of immunosuppression, which can be improved by nanotechnological approaches. Further investigations are ongoing to assess their antitumoral efficacy in other tumor models (i.e. breast and pancreatic cancer), with the final aim of clinical translation. Citation Format: Fernando Torres Andon, Alba Pensado-López, Clement Anfray, Aldo Ummarino, Iago Fernández-Mariño, Lucia Sanjurjo, Jose Crecente-Campo, Eduardo Fernández-Megía, Flavia Castro, Maria Jose Oliveira, Alfonso Calvo, Rosario García Campelo, Alberto Mantovani, Maria Jose Alonso, Paola Allavena. Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 715.

Abstract 4210: Stromal remodeling danger signals regulate “second-touch” antigenic encounters and promote CD8+ T cell effector differentiation in the TME

Abstract Tumor immune infiltration (“hot” tumor microenvironment, TME) constitutes a powerful predictor of outcomes and immunotherapy efficacy, however underlying mechanisms remain elusive. Recent evidence shows that TME-repriming of incoming CD8+ T cells is required for full effector functionality. Reminiscent of the “second-touch” hypothesis proposed by Klaus Ley for CD4+ differentiation, tumor-draining lymph node (TDLN) priming (“first-touch”) generates antigen-specific stem-like TCF1+CD8+ T cells. These stem-like CD8+ T cells then migrate to the tumor periphery where “second-touch” antigenic encounters with non-migratory CCR7neg dendritic cells (DC) (importantly, the cross-presenting cDC1 subset) leads to effector CD8+ T cell differentiation and tumor entry. These second-touch encounters occur within specialized stromal niches that are rich in antigen-presenting cells (APC). We have hypothesized that productive APC:CD8+ interactions require stroma-derived signals that emulate provisional matrix remodeling in embryonic development and/or adult wound healing. Thus, stroma remodeling by the expanding tumor translates into a conserved “danger signal” that licenses APC to activate CD8+ T cell responses. We have focused on a prototypical pathway of provisional matrix remodeling, the regulated proteolysis of the matrix proteoglycan versican (VCAN) to generate a bioactive fragment endowed with immunostimulatory activities, versikine. Stromal VCAN proteolysis at the site releasing versikine is associated with T-cell infiltration across human solid and liquid tumors. To investigate the mechanisms by which versikine regulates first- and second-touch antigenic encounters, we dissected the immune microenvironment of versikine-replete tumors of immunocompetent models at single-cell resolution. Non-migratory Ccr7neg cDC1 in versikine-replete tumors overexpress MHC-I and MHC-II antigen presentation machinery (Tap1, Ciita), maturation (Irf7, ISGs, Tlr3) and co-stimulation markers (Cd86, 4-1BBL), while Ccr7pos DC (mregDC) markedly overexpress Cd40 compared to control mregDC. In parallel, Cd4+ Tfh-like cells (overexpressing Cd40lg, Icos and Maf) accumulate in versikine-replete tumors, mirroring the accumulation of CD4+ T-cells in the stroma of human tumors displaying intense VCAN proteolysis. Consequently, versikine-replete tumors demonstrate expansion of late effector CD8+ T cells (Gzmb+Prf1+). Versikine reverses anti-PD-1 refractoriness in the “cold” model, Lewis Lung Carcinoma (LLC). In “warm” tumor models, versikine with anti-PD-L1 suffices to eradicate tumors and establish immune memory with resistance to tumor rechallenge. Our presentation will focus on the mechanistic dissection of stromal signals regulating antigen presentation across models as well as approaches for translation into novel vaccine platforms. Citation Format: Athanasios Papadas, Duncan Hong, Alexander Cicala, Alicia Gibbons, Daniel J. Lagal, Yun Huang, Yaling Dou, Fotis Asimakopoulos. Stromal remodeling danger signals regulate “second-touch” antigenic encounters and promote CD8+ T cell effector differentiation in the TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4210.

Abstract 6531: Anti-tumor immune function of NR2F6, an orphan nuclear receptor, in stromal cells

Abstract Growing evidence points to the importance of the tumor microenvironment (TME) in tumor growth control, progression, and therapy response. Among those is the greater understanding of immune system components within the TME as mediators of tumor growth control. NR2F6 is an orphan nuclear receptor, which was shown to serve as an immune intrinsic checkpoint component and has been suggested to elicit tumor-intrinsic suppression of anti-tumor immunity. Correspondingly, genetic depletion of NR2F6 in CD8 T cells abrogates the immune-activating cytokines (e.g., IL-2, IFNg). In addition, genetic ablation of NR2F6 in melanoma cells activates CD8 T cell-mediated anti-tumor immunity partly by suppressing the expression of tumor-intrinsic immune repressors NACC1 and FKBP10. Notably, combined genetic ablation of tumor cells (NR2F6 KO melanoma) and stroma cells (NR2F6 KO mouse) resulted in a more pronounced inhibition of tumor growth compared with ablation of either tumor cells or TME. The latter provides the foundation for screening NR2F6 inhibitors, which are expected to have a strong systemic (tumor inhibition and immune cell activation) effect on tumor growth. Interestingly, single-cell transcriptomic data from human tumors identified high expression of NR2F6 in stroma cells, cancer-associated fibroblasts (CAFs), and tumor-associated endothelial cells (TAEs). Consistent with these, scRNAseq analysis of mouse melanoma revealed higher expression of NR2F6 in SMA+ immune suppressive myo-CAFs than in immune activating CAFs. Studies that directly reveal the role of stromal NR2F6 in anti-tumor immunity and tumor growth and the state of NR2F6 inhibitors will be discussed. Citation Format: Hyungsoo Kim, Yongmei Feng, Marria Radaeva, Eduard Sergienko, Artem Cherkasov, Ze'ev A. Ronai. Anti-tumor immune function of NR2F6, an orphan nuclear receptor, in stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6531.

Abstract 2309: 3D spatial quantification of lymphocyte infiltration and collagen features in the tumor microenvironment using a novel assay: 3D I/O Pro™

Abstract Background: The composition of the tumor microenvironment (TME) is a major determinant of response to therapy in many solid tumors. To-date, characterization of the TME has been based on limited analysis of thin tissue sections. Here we demonstrate the utility of an end-to-end 3D spatial biology workflow, the 3D I/O Pro™, based on whole tissue imaging, to identify and quantify tumor cells, lymphocytes, and collagen features in human FFPE tissue samples. Methods: Five human colorectal cancer FFPE blocks were deparaffinized, stained with nuclear (TO-PRO-3) and general protein (eosin) dyes, and cleared using a modified iDISCO protocol. Entire samples were imaged at 2 µm/pixel resolution with a hybrid open-top light-sheet microscope, the 3Di™. Regions of interest (ROIs) with a volume of 0.5 mm3 were re-imaged at 0.33 µm/pixel. Cell nuclei and collagen were segmented and 3D spatial relationships between tumor cells, lymphocytes, and collagen fibers were quantified. Analyses were performed within select ROIs using 3Dai™ tools including U-Net, CytoMAP, CT-FIRE, CurveAlign, and custom Python scripts. Results: The ratio of stromal to tumor parenchymal lymphocytes (lymphocyte infiltration ratio) varied from 1.4 up to 9.1 in 3D volumes and 1 to 25 in 2D virtual sections taken throughout all 5 CRC samples. The ratio of the collagen fibers within 100 µm of the tumor border-oriented perpendicular to the tumor-stromal boundary compared to those oriented parallel to the tumor-stromal boundary was 0.37 in a proof-of-concept evaluation of a 2D virtual section with a lymphocyte infiltration ratio of 12. Qualitatively, areas with perpendicular collagen had more lymphocyte infiltration into the tumor parenchyma than areas with parallel collagen orientation. Conclusions: We demonstrated that the 3D I/O Pro™ pipeline can quantify lymphocyte density in tumor parenchyma and stroma and analyze collagen features, including orientation, within 3D ROIs in the TME. This workflow allows us to characterize tumors based on many complex spatial relationships and could have broad applicability in research and development of novel cancer therapies that target tumor fibrosis or other features of the TME. In the future, we plan to correlate features from the 3D TME with response to immunotherapy and use these features to refine histologic definitions of immune spatial phenotypes. Citation Format: Caleb Stoltzfus, Jasmine Wilson, Nathan Grant, Alexandra Alvarsson, Anna DeWitt, David Simmons, Brandy Olin, Bonnie Phillips, Laura A. Dillon, G. Travis Clifton, Nicholas P. Reder. 3D spatial quantification of lymphocyte infiltration and collagen features in the tumor microenvironment using a novel assay: 3D I/O Pro™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2309.

Abstract 1488: Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer associated with high rates of recurrence and metastasis, which contribute to poor overall survival. The challenges in treating TNBC stem from its high intratumor heterogeneity and lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 druggable targets. The tumor microenvironment (TME) plays a crucial role in disease progression. Within the TME, complex spatial interactions among tumor cells, immune cells, stroma, angiogenesis- and hypoxia-inducing factors collectively contribute to the advancement and spread of TNBC. In-depth understanding of the TME and interactions of the various components within can elucidate newer therapeutic targets, enhancing the prognosis of TNBC patients. Imaging mass cytometry (IMC) enables detailed analysis of cell-cell and cell-matrix interactions within the TME at the single-cell level. In this study, we aim to characterize the spatial relationships among various cell types within the TNBC TME using IMC, while also investigating differences in the TME of TNBC patients who developed distant metastasis (DM) compared to those who remained non-metastatic (NM). IMC was performed on formalin-fixed paraffin-embedded TNBC primary resection tissues (n= 16; DM= 7, NM= 9) from Stavanger University Hospital, Norway, using a panel of 25 metal-conjugated antibodies. Regions of interest were marked by the pathologist and the slides were laser ablated using Hyperion Imaging System. DeepCell was used to segment single cells based on surface markers. Mean intensity values of all markers for each cell were extracted and consolidated in R scripts for unsupervised clustering. Cell clusters were annotated based on phenotypic markers. Normalized expression of markers was compared for each cell type across DM and NM patients, including markers for cell proliferation, angiogenesis, hypoxia, and cell migration. Neighborhood analysis examined spatial associations between different cell types, highlighting differences in DM and NM patients. The IMC panel successfully provided spatial insights into the TME, including the characterization of tumor cells, immune cells (B cells, T cells, macrophages), endothelial cells, and proliferating cells. We identified 18 unique phenotypic cell clusters that were present at different frequencies in DM and NM patients. These clusters demonstrated different expression profiles of functional markers. t-SNE plots revealed distinct patterns of cell type distribution and unique protein expression profiles, highlighting significant differences between the two groups. Our data suggest a distinct spatial organization of the TME in TNBC patients who develop metastasis. This warrants further investigations to determine the role and interplay of TME features in predicting the risk of distant metastasis in TNBC. Citation Format: Mahak Bhargava, Jaspreet Kaur, Geetanjali Saini, Junjun Zheng, Yitian Xu, Shu-Hsia Chen, Ping-Ying Pan, Emma Rewcastle, Umay Kiraz, Emiel Adrianus Janssen, Ritu Aneja. Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1488.

Abstract 3574: Cancer testis antigen expression correlates with stromal natural killer cell activation and longer overall survival in small bowel neuroendocrine tumors

Abstract Background: Patients with small bowel neuroendocrine tumors (SBNET) frequently present with metastatic disease and the efficacy of available therapies, including immune checkpoint blockade (ICB), is limited. Toward developing novel immunomodulatory strategies, we interrogated the tumor immune microenvironment of SBNETs using bulk transcriptional and digital spatial profiling (DSP). Methods: Patients with SBNET who underwent resection at MD Anderson Cancer Center from 2003-2016 were retrospectively analyzed. Overall survival (OS) was assessed using the Kaplan-Meier method and the Cox proportional hazards model was used for multivariate analysis (MVA). Bulk transcriptional profiling was performed using the Nanostring PanCancer-Immune Panel. DSP with whole human transcriptome was performed using PanCK to segment tumor and adjacent stroma; 88 distinct regions were analyzed using differential gene expression and Reactome pathway enrichment pipelines. Results: Resected SBNET from 42 patients were selected for transcriptional analysis. Unsupervised clustering of gene expression demonstrated dichotomization into high and low cancer testis antigen (CTA) expression. CTA-high patients (n=12) demonstrated elevated expression of type-I interferons (IFN), and CTAs such as PRAME, GAGE1 and MAGEA3 and had significantly longer OS compared to CTA-low patients (n=30; median OS not reached vs 1975 days, p=0.008). MVA controlling for age, sex, stage, and Ki-67% confirmed CTA-high status as an independent predictor of longer OS (hazard ratio [HR] 0.2, 95% confidence interval [CI] 0.05-0.83, p=0.014). A pilot DSP experiment on 4 CTA-high and 4 CTA-low tumors confirmed differential gene expression in the PanCK+ tumor compartment of these samples (R2=0.09, p=0.001). In particular, epigenetic modification genes (e.g., H2BC8, H4C8, H3C1, p<0.001) and epigenetic pathways (e.g., DNA methylation, histone acetylation) were enriched in CTA-high tumors. CTA-high tumors exhibited increased M2 macrophage presence (p=0.025) and increased NK cell activation within the PanCK- stromal compartment (p=0.001). Conclusions: High CTA and type-I IFN expression in resected SBNET identifies patients with longer survival, agnostic of stage or grade. While CTA expression has been implicated in tumor immunogenicity, this is the first work to identify a clinically relevant signal in SBNET. Increased IFN in CTA-high tumors, corroborated by increased stromal NK cell activation, suggests that enhanced immunogenicity may drive this survival difference. Spatial transcriptomic analysis reveals epigenetic differences in CTA-high tumors and highlights the potential for combination epigenetic modifiers and immunotherapy in future trials. Citation Format: Reed I. Ayabe, Yongwoo D. Seo, Brenda Melendez, Rossana Lazcano, Brittany C. Fields, Khalida Wani, Sarah Johnson, Manoj Chelvanambi, Courtney Hudgens, Sharia D. Hernandez, Nadim J. Ajami, Jennifer A. Wargo, Alexander J. Lazar, Daniel M. Halperin, Jeannelyn S. Estrella, Jessica E. Maxwell. Cancer testis antigen expression correlates with stromal natural killer cell activation and longer overall survival in small bowel neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3574.

Abstract 1580: Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancers with a survival rate of less than 12% at 5 years. It is estimated that around 14% of human PDAC contain mutations in genes involved in DNA Damage Repair (DDR), including BRCA1/2, ATM, and others. Additionally, PDAC has an extensive network of extracellular matrix components surrounding the tumor in the stroma, such as cancer-associated fibroblasts, which promote cancer cell growth. The interactions of the tumor with its tumor microenvironment and stroma can bring vulnerabilities that could be targeted if identified. New therapeutic strategies to those targets can be developed for specifically PDAC with DDR mutations. Using publicly available RNA-seq dataset of PDAC patients from The Cancer Genome Atlas (TCGA), I computationally estimated the infiltration of immune and stromal cell types into the tumor. From the analyses, I found that there was a significant difference in the migration of Common Lymphoid Progenitor (CLP) cells into PDAC with mutations to DDR genes. CLP cells usually divide into T-cells, B-cells, and NK cells, suggesting that DDR mutations could cause a more immunogenic environment. There was also a decrease in the infiltration of stromal elements, mainly fibroblasts, into PDAC with mutations to DDR genes. In the experiment I am currently working on, I hypothesize there are also in vivo differences in the infiltration rates of certain immune cell types and fibroblasts into BRCA2-mutated PDAC, since BRCA2 is the most commonly mutated DDR gene in PDAC. For my current experimental design, the mice in the experimental group will be orthotopically implanted with PDAC with Brca2 knockout. The control group will have mice orthotopically implanted with PDAC without Brca2 knockout. After letting the tumors grow for 3 weeks, the mice will be sacrificed and the pancreatic tumors will be collected and stained through trichrome staining. Trichrome staining probes for the stromal cancer-associated fibroblasts. Furthermore, immunohistochemistry (IHC) staining will be done to look for specific populations of cells, such as specific types of fibroblasts, effective and immunosuppressive immune cells including T-cells, B-cells, and NK cells. The aim is to see whether the computational findings in regards to the tumor microenvironment are also reflected in vivo. I will have the data from this experiment by the time of the conference in April 2024. Overall, my project is about investigating the tumor microenvironment and fibroblast abundance in PDAC with BRCA2 knockout mice, using computational data, IHC staining and trichrome staining. Citation Format: Fatimah Al-Musawi, Chang-il Hwang. Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1580.

Abstract 914: Combination analysis of tumor-associated collagen frameworks and tumor immune phenotype of lung carcinomas using virtual staining

Abstract An emerging predictive parameter of immunotherapy response is the patient’s tumor immune status, generally classified as “inflamed”, "immune excluded” and “desert”. Historically, classification was performed semi-quantitatively with somewhat subjective parameters as evidenced by the recent Delphi Workshop consensus. There are a variety of approaches to classifying immune status, most based upon analysis of H&E images (counting tumor infiltrating lymphocytes, TILs), or counting subpopulations of immune cells using immunologic staining. However, use of multiplexed or multiple stains has recently been explored to better quantify the counts and types of immune cells in individual tissue compartments. While the immune microenvironment represents an important parameter in predicting patient response, other regions such as the extracellular matrix (ECM) may yield complementary information, as collagen-rich ECMs may present a barrier to drug diffusion and the orientation of fibers may direct the migration of malignant cells. Here we present a proof of concept virtual staining enhanced image analysis pipeline, which converts autofluorescence signals from a single tissue section into virtual H&E and Masson’s Trichrome along with virtual detection of pan cytokeratin (AE1/AE3/PCK26) and CD45 (LCA) positive cells. We apply image analysis to the panel of virtual stains to study the spatial and case-by-case heterogeneity of tumor collagen frameworks and immune phenotype. Using a standard slide scanner (Axio Scan Z1, Zeiss), multiple autofluorescence images were captured from unstained sections (4-6um thick) of lung tissue. The virtual staining was performed by four deep neural networks trained in a supervised learning fashion. Select chemical stains were performed on previously scanned tissues and reviewed by pathologists side-by-side with virtual stains to ensure consistency and quality control. Four perfectly registered WSI virtual stain images were generated from each tissue section. The multi-stain results were rendered from a variety of lung cancers, including tissue microarray slides. Image analysis was performed using HALO (Indica Labs) and custom python scripts. We identified unique areas of tumor and adjacent stroma with heterogeneous immune phenotype and collagen characteristics, suggesting that an interplay might exist that could be utilized to improve patient selection or prognosis when deploying advanced AI tools. Future work includes applying this virtual stain technique retrospectively to cases with known immunotherapy treatment responses to determine the prognostic significance of the combined immune/ECM phenotype. Citation Format: Serge Alexanian, Yair Rivenson, Ning Xuan, Brian Cone, Zihang Fang, Sean Meyering, Luis A. Carvalho, David Palacios, Raymond Kozikowski. Combination analysis of tumor-associated collagen frameworks and tumor immune phenotype of lung carcinomas using virtual staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 914.

Abstract 6180: Tumor microenvironment phenotypes predict recurrence in African Americans diagnosed with colorectal cancer: A comparative study of manual grading with digital pathology

Abstract African Americans face a higher rate of colorectal cancer (CRC) incidence and mortality compared to other ethnicities. Identifying key prognostic factors is essential for better outcomes in this group. This study focused on the prognostic value of stromal grading in the tumor microenvironment (TME) of African American CRC patients, using both manual grading and digital pathology. A retrospective analysis of CRC cases in African Americans was conducted. Two cohorts were established: a control cohort (patients without recurrence) and a recurrence cohort (patients with recurrence). Stromal grading was performed manually, categorizing tumors into Grade 1 (low risk), Grade 2 (moderate risk), and Grade 3 (high risk), and we also quantified the TME percentages by digital pathology: myxoid, collagen, immune and tumor. In the control cohort (N=40), stromal grading identified 30 Grade 1, 7 Grade 2, and 3 Grade 3 cases. Conversely, the recurrence cohort (N=17) demonstrated a starkly different distribution, with 1 Grade 1, 3 Grade 2, and 13 Grade 3 cases. The Chi-square statistic was notably significant (p<0.0001), indicating a strong association between higher stromal grades and the likelihood of recurrence. By unpaired t-test, high myxoid stroma (p<0.0001), low collagen (p<0.0001), and low tumor (p=0.0002) by digital pathology were associated with recurrence. TME phenotypes are a significant prognostic and recurrence indicator in African American patients with CRC. The marked difference in stromal grades and digital pathology features between the control and recurrence cohorts suggests that higher stromal grades, along with higher myxoid, lower collagen and lower tumor percentages are associated with an increased risk of recurrence. These findings underscore the need for tailored therapeutic strategies and more vigilant surveillance in African American patients exhibiting high-risk TME phenotypes. The efficacy of manual and digital pathology evaluation of the TME also warrants further exploration to enhance prognostic accuracy and treatment personalization. Citation Format: Dongling Wu, Rong Xia, Mouyed Alawad, Lili Lee, Tamas Gonda, Wenqing Cao, Sean Hacking. Tumor microenvironment phenotypes predict recurrence in African Americans diagnosed with colorectal cancer: A comparative study of manual grading with digital pathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6180.

Abstract 73: Spatial profiling of pediatric rhabdomyosarcoma to elucidate their immunosuppressive tumor microenvironment

Abstract Background: Pediatric rhabdomyosarcoma (RMS) is a mesenchymal soft tissue malignancy. Despite intensive chemotherapy regimens, there is currently a gap in improving treatment outcomes for RMS. Chemotherapy can modulate the tumor microenvironment (TME) by altering the immune cell composition or their functions. However, the impact of chemotherapy in pediatric RMS TME is unclear. RMS tumors are also considered as “cold” tumors due to low immune infiltration, which could be a determinant of why immunotherapy is unlikely to work. Additional investigations are required to determine the reasons of why RMS is “cold” and whether a combined immunotherapy and chemotherapy approach should be administered for this hard-to-cure cancer. Methods We conducted for the first time spatial GeoMx whole transcriptome atlas and 51-plex Akoya CODEX profiling for a tissue microarray (TMA) from a cohort of pediatric RMS patients (n = 12) with pre-chemotherapy (n = 5) and post-chemotherapy (n = 5) samples. Regions of interest (ROIs) were selected based on tumor areas with immune cell infiltration, with the lymphocytes, tumor cells, and nuclei visualized using fluorescent morphological markers CD45, Desmin, and DAPI. ROIs were segmented into tumor stroma compartments and immune cell compartments. Computational analyses were conducted on the transcriptomics data using R package standR including quality control, normalization, differential expression analysis, gene set enrichment analysis, and cell type deconvolution. For CODEX data, we preformed cell segmentation, followed by further quality control, cell phenotyping, cellular neighborhood and cellular interaction analysis. Results: From differential expression analysis of the immune compartment, we have identified type I interferon-related genes (DTX3L, ADAP2, RNF135) and reactive oxygen species (ROS)-induced gene (NCF1) are upregulated, and an antioxidant enzyme gene (PRDX2) is downregulated in post-chemotherapy samples compared to pre-chemotherapy samples. Gene set enrichment analysis of the immune compartment revealed an enriched antigen presentation pathway in post-chemotherapy samples. In the tumor stroma compartment, we identified increased TGF-β binding pathway in post-chemotherapy samples. Cellular deconvolution revealed a higher proportion of endothelial cells in post-chemotherapy samples, which is validated via CODEX imaging analysis. Cellular neighborhoods and cell-cell interaction analysis for CODEX data also reveal how chemotherapy affect functional units and cellular interactions in RMS TME with spatial context preserved. Conclusion: By applying spatial transcriptomic and multiplexed imaging to RMS samples, we have revealed key genes, pathways and cell types involved in chemotherapy-induced alterations in the TME. This will provide a new perspective for developing novel chemoimmunotherapy for RMS patients. Citation Format: Cui Tu, Chin Wee Tan, Ning Liu, Arutha Kulasinghe, Fernando Souza-Fonseca-Guimaraes. Spatial profiling of pediatric rhabdomyosarcoma to elucidate their immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 73.

Abstract 726: The retinoid X receptor agonist MSU42011 enhances efficacy of anti-PD1 therapy in a HER2+ breast cancer model through a CD4-IL12 axis

Abstract Immunotherapy is rarely effective in breast cancer and most current therapeutic options fail to target the stromal compartment. We have developed a new small molecule, MSU42011, that induces tumor regression in experimental models of Kras-driven lung cancer and HER2+ breast cancer. This tumor regression is dependent on a functional immune system. Here, we dissected the tumor stroma, revealing a CD4 and IL12a dependency for the anti-tumor activity of MSU42011. Cancer immunotherapy responses are mostly driven by CD8 T cells. Using a murine HER2+ tumor model, anti-CD8 antibodies (200ug/mouse) were injected 24 hours before treatment with MSU42011 (100MG/KG in power diet) was started. Mice were maintained on diet for 10 days. Anti-CD8 antibodies were also administered on days 3, 7 and 10. The combination did not affect the inhibition of tumor growth by MSU42011, suggesting that its anti-tumor efficacy is not dependent on CD8 T cells. In contrast, tumors treated with MSU42011 and anti-CD4 antibodies grew (p=0.0016 versus MSU42011 alone) at the same rate as tumors treated with control diet (p=0.0139 versus MS42011). Furthermore, anti-CD4 reversed inhibition of tumor growth by MSU42011 in a carcinogen-induced lung tumor model. Interestingly, MSU42011 increased the levels of IL12 by 4x (p=0.01) and IFN by 8x (p=0.03) compared to the controls. These data suggest that MSU42011 increased Th1 CD4 T cell polarization; the increase in IL12 was lost following CD4 T cell depletion. Following depletion of IL12a with antibodies (loading dose of 1 mg, followed by 0.5mg per mouse IP), tumors treated with the combination of MSU42011 and anti-IL12a antibodies grew at the same rate as the controls. T cell engagement with MSU42011 suggests that this small molecule is a good candidate for combination with immunotherapy. HER2+ MMTV-Neu mice with established tumors were treated with the combination of MSU42011 and anti-PD1 antibodies. Treatment with MSU42011 at 100mg/Kg of diet for 10 days, followed by anti-PD1 antibodies (200ug) twice weekly failed to increase survival. In contrast, 2 doses of anti-PD1 antibodies (300ug), followed by treatment with MSU42011 increased survival by 10 days compared to controls. The tumors in the MSU42011+anti-PD1 group initially grew rapidly, with increased redness and heat, suggesting an enhanced inflammatory response. When anti-PD1 antibodies were given at a lower loading dose (200ug and then 100ug twice weekly) followed by a higher dose of MSU42011 (300 mg/Kg of diet), survival markedly increased (p=0.052). These findings demonstrate that activation of RXR in the stroma can be used to increase the effectiveness of immunotherapy. Citation Format: Ana Sofia Leal, Karen T. Liby. The retinoid X receptor agonist MSU42011 enhances efficacy of anti-PD1 therapy in a HER2+ breast cancer model through a CD4-IL12 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 726.

Abstract 76: Correlated multi-scale 3D optical microscopy of tumor tissues

Abstract Three-dimensional (3D) optical microscopy in combination with advanced tissue clearing methods permits the interrogation of intact large-sized tumor tissues. There are many optical microscopy methods (e.g. light-sheet and confocal microscopy) offering 3D tumor images at varying levels of spatial resolution (e.g. organ, tissue, cell, and molecule level). However, it is a challenge to correlatively integrate various resolution 3D images obtained by different optical microscopy of tumor tissue. In this project, we developed a correlated multi-scale 3D optical microscopy method that enables the acquisition of macroscopic to microscopic spatial information from a single tumor tissue in 3D. Firstly, we used light sheet microscopy to image a whole large-sized, cleared tumor tissue and analyzed the results to identify regions of interests (ROIs). After defining ROIs in the 3D whole tumor image, we added a UV-activatable visible dye, spiropyran, in the agarose gel surrounding the cleared tumor and exposed the sample to a UV light sheet horizontally at the height (z-axis) of the tumor where the ROIs present. The activated dye showed ‘purple’ colored lines in the agarose gel and we physically marked the gel along the visible colored lines. We further sectioned the tumor at the ROI positions using a vibratome after reversing tissue clearing process. After immunofluorescence staining and aqueous-based tissue clearing, confocal microscopy allowed the localization of various cell types in the ROIs of tumor macrosections. To validate the method for multi-scale evaluation of cancer immunotherapy, we tested it with control and DMXAA-treated mouse mammary tumor tissues. We successfully acquired the 3D images of whole tumors by capturing autofluorescence signals with a 633 nm excitation laser and its matched emission filter. Based on the autofluorescence tumor images, we defined two ROIs for each control and treated tumor and performed optical marking and physical sectioning of the tumors as described above. Next, we stained the macrosections (400 µm thick) with DAPI, CK8-DL488, CD45-DL550, and ER-TR7-DL680 to visualize cancer, immune, and stromal cells in the ROIs at cellular resolution. We quantitatively analyzed the correlated multi-scale 3D image data for assessment of the effects of DMXAA treatment on the tumor microenvironment including eradiation of cancer cells, immune infiltration, and remodeling of tumor stroma. In summary, this new microscopy method will provide comprehensive spatial information of tumor tissues and benefit a broad cancer research field such as the discovery of new tumor biomarkers and the development of effective cancer therapies. Citation Format: Jingtian Zheng, Steve Seung-Young Lee. Correlated multi-scale 3D optical microscopy of tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 76.

Abstract 290: The role of tumor stromal topography-regulated genes in modulating the cellular landscape of tumor stroma in breast cancer

Abstract The tumor remodels the surrounding extracellular matrix fibers to become topographically aligned at the tumor front, further facilitating cancer cell migration. Our recent studies show that aligned tumor stromal topography promotes the detachment of breast cancer cells from the main tumor, then, in the following cell migration, upregulating genes such as genes encoding the cytochrome P450 family 1 subfamily A member 1 (CYP1) and inhibitor of DNA binding (ID) proteins. In this study, we further explored the role of topography-regulated genes in modulating the cellular landscape of tumor stroma. By analyzing the correlation of topography-regulated genes with stromal cell infiltration in bioinformatics databases, we found significant positive correlations between the RNA expression levels of most topography-regulated genes and the infiltration levels of cancer-associated fibroblasts and endothelial cells. Notably, the strongest correlation was between the expression level of ID3 and the infiltration levels of cancer-associated fibroblasts (r = 0.509) and endothelial cells (r = 0.506). Additionally, ID3 remained highly expressed in circulating tumor cells but exhibited low expression at distant metastatic sites. These findings suggest that genes regulated by tumor stromal topography, especially ID3, may play a role in neovascularization and fibrosis of the tumor microenvironment and the survival adaptation of cancer cells in circulation, thereby promoting metastasis. Citation Format: Chia-Yi Su. The role of tumor stromal topography-regulated genes in modulating the cellular landscape of tumor stroma in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 290.

Abstract 5398: Single-cell transcriptomic analysis of partial endothelial-to-mesenchymal transition in sprouting angiogenesis

Abstract Endothelial-to-mesenchymal transition (EndoMT) is a process of morphological, functional, and molecular shift of endothelial cells (EC) towards a mesenchymal identity. This process is critical for embryonic development, and knockout animals lacking copies of the individual master EndoMT transcription factors – including Slug, Snail, and Twist – exhibit significant cardiovascular defects. We previously showed that while Slug knockout animals have a modest developmental vascular phenotype, there is a striking loss of tumor angiogenesis in these animals. Based on these and other data, we hypothesize that sprouting angiogenesis involves transient acquisition of an intermediate state of the EndoMT program –i.e., partial EndoMT (pEndoMT). To test this hypothesis, we used single cell RNA sequencing at day 2 and day 6 following TGF-β activation to map the transcriptomic changes associated with complete endothelial-to-mesenchymal transition. In parallel, we sequenced EC activated to undergo sprouting angiogenesis in vitro at the same timepoints. We show that angiogenic EC transiently acquire a transcriptomic profile of the pEndoMT state, but then later reacquire the transcriptomic profile of committed EC rather than transition completely into mesenchymal cells. We further use this dataset to identify several candidate genes that appear to be regulated by Slug and associated with the pEndoMT state. Future work will use in vitro and in vivo angiogenesis models to identify how pEndoMT is regulated in angiogenesis, and how this regulation may differ in the presence of tumor stroma. Citation Format: Kapil Thapa, Christopher J. Hatch, Christopher Hughes, Jennifer S. Fang. Single-cell transcriptomic analysis of partial endothelial-to-mesenchymal transition in sprouting angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5398.

Abstract 4274: Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach

Abstract Background: The tumor microenvironment (TME) plays a crucial role in driving tumor development. Among the constituents of the tumor stroma, cancer-associated fibroblasts (CAFs) are a significant component. CAFs are actively involved in tumor progression by modulating the architecture of the TME through increased deposition of various collagens resulting in tumor fibrosis. Several studies have shown that CAFs have heterogeneity within, and between, individual tissues. TGF-β is thought to be the main driver of tumor fibrosis, however, the field lacks a characterization of the specific collagen deposition of CAFs from different tissues. In this study, we investigated the fibrotic activity of CAFs from various tissues by measuring the production of three specific collagen peptides in vitro by use of non-invasive clinically validated biomarkers. Methods: Primary human CAFs from pancreas (pCAF), colon (cCAF), breast (bCAF) and lung (lCAF) were cultured over a 12-day period in ficoll-based media under both unstimulated conditions and TGF-β stimulation. Additionally, cells were subjected to treatment with the ALK5/TGF-β1 receptor kinase inhibitor (ALK5i). The assessment of collagen I (PRO-C1), collagen III (PRO-C3), and collagen VI (PRO-C6) formation was conducted in the cell supernatant at day 3, day 6, day 9, and day 12 using competitive enzyme-linked immunosorbent assay (ELISA). Results: For lCAFs, cCAFs, and pCAFs, the addition of TGF-β led to a notable elevation in the levels of PRO-C1, PRO-C3, and PRO-C6 within the supernatant. Conversely, in the case of bCAFs, stimulation with TGF-β did not elicit remarkable alterations in the levels of PRO-C1 and PRO-C3 compared to unstimulated cells. Notably, bCAFs exhibit elevated intrinsic activity (high PRO-C3) even in the absence of TGF-β stimulation whereas lCAFs and cCAFs exhibited relatively low level of PRO-C3. Additionally, across all different CAFs, the introduction of ALK5 inhibitor (ALK5i) to the culture medium resulted in a considerable reduction in PRO-C1, PRO-C3, and PRO-C6 levels, returning them to the baseline of the unstimulated control or even lower. Distinct collagen formation patterns were observed among the various CAFs. CAFs derived from breast cancer exhibited the highest release of PRO-C1 and PRO-C3, while CAFs isolated from colon cancer displayed the highest levels of PRO-C6. Conclusion: These findings underscore the heterogeneity in collagen production among different CAFs, providing valuable insights into the extracellular matrix dynamics within distinct tumor microenvironments. Moreover, non-invasive biomarkers demonstrate the capability to differentiate between different CAF subtypes. Lastly, our model proves to be a valuable tool for anti-fibrotic drug screening, as evidenced by the consistent suppression of TGF-β1 signaling and reduced levels of PRO-C1, PRO-C3, and PRO-C6 upon treatment with ALK5i across all cases. Citation Format: Annika Hettich, Neel I. Nissen, Morten A. Karsdal, Nicholas Willumsen. Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4274.