Cancer Stromal Cells Research Articles

EphA-Mediated Regulation of Stomatin Expression in Prostate Cancer Cells.

Tumor growth and progression are affected by interactions between tumor cells and stromal cells within the tumor microenvironment. We previously showed that the expression of an integral membrane protein, called stomatin, was increased in cancer cells following their association with stromal cells. Additionally, stomatin impaired the Akt signaling pathway to suppress tumor growth. However, it remains unclear how stomatin expression is regulated. To explore this, we examined the cell surface molecules that can transduce the intercellular communication signals between cancer cells and stromal cells. Among these molecules, EphA3 and EphA7 receptors and their ligand ephrin-A5 were found to be expressed in prostate cancer cells, but not in prostate stromal cells. Cell-to-cell contact of prostate cancer cells through the EphA-ephrin-A interaction suppressed stomatin expression, while knockdown of EphA3/7 or ephrin-A5 increased stomatin expression. This increase contributed to an inhibition of prostate cancer cell proliferation. Intracellularly, the binding of ephrin-A to EphA attenuated extracellular signaling-regulated kinase (ERK) activation that promoted stomatin expression. Furthermore, ELK1 and ELK4, which are Ets family transcription factors phosphorylated by ERK, were involved in the induction of stomatin expression. We also found that higher Gleason score prostate cancer tissue samples had increased activation of EphA, while the stomatin expression and activated ERK and ELK levels were all low. In the mouse xenograft tumor samples generated by implantation of prostate cancer cells, EphA3 phosphorylation was attenuated and the ERK-ELK signaling and stomatin expression were enhanced in the area where stromal cells infiltrated the tumor. The EphA-mediated signaling suppresses the ERK-ELK pathway, leading to the reduction of stomatin expression that affects prostate cancer malignancy.

Multiparameter imaging reveals clinically relevant cancer cell-stroma interaction dynamics in head and neck cancer.

Epithelial tumors are characterized by abundant inter- and intra-tumor heterogeneity, which complicates diagnostics and treatment. The contribution of cancer-stroma interactions to this heterogeneity is poorly understood. Here, we report a paradigm to quantify phenotypic diversity in head and neck squamous cell carcinoma (HNSCC) with single-cell resolution. By combining cell-state markers with morphological features, we identify phenotypic signatures that correlate with clinical features, including metastasis and recurrence. Integration of tumor and stromal signatures reveals that partial epithelial-mesenchymal transition (pEMT) renders disease outcome highly sensitive to stromal composition, generating a strong prognostic and predictive signature. Spatial transcriptomics and subsequent analyses of cancer spheroid dynamics identify the cancer-associated fibroblast-pEMT axis as a nexus for intercompartmental signaling that reprograms pEMT cells into an invasive phenotype. Taken together, we establish a paradigm to identify clinically relevant tumor phenotypes and discover a cell-state-dependent interplay between stromal and epithelial compartments that drives cancer aggression.

Metabolic regulation of the tumour and its microenvironment: The role of Epstein-Barr virus.

The Epstein-Barr virus (EBV), the first identified human tumour virus, infects over 95% of the individuals globally and has the potential to induce different types of cancers. It is increasingly recognised that EBV infection not only alters cellular metabolism, contributing to neoplastic transformation, but also utilises several non-cell autonomous mechanisms to shape the metabolic milieu in the tumour microenvironment (TME) and its constituent stromal and immune cells. In this review, we explore how EBV modulates metabolism to shape the interactions between cancer cells, stromal cells, and immune cells within a hypoxic and acidic TME. We highlight how metabolites resulting from EBV infection act as paracrine factors to regulate the TME, and how targeting them can disrupt barriers to immunotherapy.

Impact of stroma remodeling on forces experienced by cancer cells and stromal cells within a pancreatic tumor tissue

Remodeling (re-engineering) of a tumor’s stroma has been shown to improve the efficacy of anti-tumor therapies, without destroying the stroma. Even though it still remains unclear which stromal component/-s and what characteristics hinder the reach of nanoparticles deep into cancer cells, we hypothesis that mechanisms behind stroma’s resistance to the penetration of nanoparticles rely heavily on extrinsic mechanical forces on stromal cells and cancer cells. Our hypothesis has been formulated on the basis of our previous study which has shown that changes in extracellular matrix (ECM) stiffness with tumor growth influence stresses exerted on fibroblasts and cancer cells, and that malignant cancer cells generate higher stresses on their stroma. This study attempts to establish a distinct identification of the components’ remodeling on the distribution and magnitude of stress within a tumor tissue which ultimately will impact the resistance of stroma to treatment. In this study, our objective is to construct a three-dimensional in silico model of a pancreas tumor tissue consisting of cancer cells, stromal cells, and ECM to determine how stromal remodeling alters the stresses distribution and magnitude within the pancreas tumor tissue. Our results show that changes in mechanical properties of ECM significantly alter the magnitude and distribution of stresses within the pancreas tumor tissue. Our results revealed that these stresses are more sensitive to ECM properties as we see the stresses reaching to a maximum of 22,000 Pa for softer ECM with Young’s modulus of 250 Pa. The stress distribution and magnitude within the pancreas tumor tissue does not show high sensitivity to the changes in mechanical properties of stromal cells surrounding stiffer cancer cells (PANC-1 with Young’s modulus of 2400 Pa). However, softer cancer cells (MIA-PaCa-2 with (Young’s modulus of 500 Pa) increase the stresses experienced by stiffer stromal cells and for stiffer ECM. By providing a unique platform to dissect and quantify the impact of individual stromal components on the stress distribution within a tumor tissue, this study serves as an important first step in understanding of which stromal components are vital for an efficient remodeling. This knowledge will be leveraged to overcome a tumor’s resistance against the penetration of nanoparticles on a per-patient basis.

Pathological examination of factors involved in PD-L1 expression in patients with oral tongue squamous cell carcinoma

BackgroundTumor tissues comprise cancer cells and stromal cells, and their interactions form the cancer microenvironment. Therefore, treatments targeting cells other than cancer cells are also actively being developed, and among them, treatment targeting PD-1, an immune checkpoint molecule that is important in tumor immune evasion, has also been indicated for head and neck cancer. PD-L1, a ligand of PD-1, is expressed in both tumor cells and stromal cells, and the scoring system based on the combined positivity rates of both types of cells, the combined positive score (CPS), is used for predicting treatment effect. However, much is unknown regarding the expression of PD-L1. In this study, we histopathologically examined factors controlling the expression of PD-1/PD-L1. This study included 37 patients who underwent resection surgery for tongue squamous cell carcinoma in the Department of Oral and Maxillofacial Surgery at Tokyo Dental College Suidobashi Hospital. The expression levels of PD-L1, α-SMA, and p53 were assessed by immunohistochemical staining.ResultsSeven participants had CPS ≥ 20, twenty-four participants had 1 ≤ CPS < 20, and six participants had CPS < 1. The overall positivity rate of α-SMA, a marker for cancer-associated fibroblasts (CAFs), was 27% (10/37 participants), and the positivity rates of α-SMA for the three CPS groups were 85.7% (6/7 participants), 16.7% (4/24 participants), and 0% (0/6 participants), respectively. In addition, the overall positivity rate of p53 was 37.8% (14/37 participants), and the positivity rates of p53 for the three CPS groups were 71.4% (5/7 participants), 37.5% (9/24 participants), and 0% (0/6 participants), respectively.ConclusionsThe expression of PD-L1 demonstrated an association with α-SMA and p53 positivity. In addition, compared with the expression of p53, the expression of α-SMA demonstrated a higher association with PD-L1 expression in patients with a high CPS. The abovementioned findings suggest that the interactions between CAFs, cancer cells, and immunocompetent cells may regulate the expression of PD-L1.

Exosome Transfer Between Different Co-implanted Human Pancreatic Cancer Cell Lines Occurs in the Primary Tumor and Multiple Metastatic Sites of Nude Mice But Not in Co-Culture In Vitro.

Exosome exchange between cancer cells or between cancer and stromal cells is involved in cancer metastasis. We have previously developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct fluorescent genetic reporters to demonstrate the role of exosomes in metastasis. In the present study, we studied exosome transfer between different pancreatic-cancer cell lines in vivo and in vitro and its potential role in metastasis. Human pancreatic-cancer cell lines AsPC-1 and MiaPaCa-2 were used in the present study. AsPC-1 cells contain a genetic exosome reporter gene labeled with green fluorescent protein (pCT-CD63-GFP) and MiaPaCa-2 cells express red fluorescent protein (RFP). Both cell lines were co-injected into the spleen of nude mice (n=5) to further study the role of exosome exchange in metastasis. Three weeks later mice were sacrificed and tumors at the primary and metastatic sites were cultured and observed by confocal fluorescence microscopy for exosome transfer. The primary tumor formed in the spleen and metastasized to the liver, as observed macroscopically. Cells were cultured from the spleen, liver, lung, bone marrow and ascites. Transfer of exosomes from AsPC-1 to MiaPaCa-2 was demonstrated in the cultured cells by confocal fluorescence microscopy. Moreover, cell fusion was also observed along with exosome transfer. Exosome transfer did not occur during in vitro co-culture between the two pancreatic-cancer cell lines, suggesting a role of the tumor microenvironment (TME) in exosome transfer. The transfer of exosomes between different pancreatic-cancer cell lines was observed during primary-tumor and metastatic growth in nude mice. This cell-cell communication might be a trigger of cell fusion and promotion of cancer metastasis. Exosome transfer between the two pancreatic-cancer cell lines appears to be facilitated by the TME, as it did not occur during in vitro co-culture.

Uncovering Porphyrin Accumulation in the Tumor Microenvironment.

Heme, an iron-containing tetrapyrrole, is essential in almost all organisms. Heme biosynthesis needs to be precisely regulated particularly given the potential cytotoxicity of protoporphyrin IX, the intermediate preceding heme formation. Here, we report on the porphyrin intermediate accumulation within the tumor microenvironment (TME), which we propose to result from dysregulation of heme biosynthesis concomitant with an enhanced cancer survival dependence on mid-step genes, a process we recently termed "Porphyrin Overdrive". Specifically, porphyrins build up in both lung cancer cells and stromal cells in the TME. Within the TME's stromal cells, evidence supports cancer-associated fibroblasts (CAFs) actively producing porphyrins through an imbalanced pathway. Conversely, normal tissues exhibit no porphyrin accumulation, and CAFs deprived of tumor cease porphyrin overproduction, indicating that both cancer and tumor-stromal porphyrin overproduction is confined to the cancer-specific tissue niche. The clinical relevance of our findings is implied by establishing a correlation between imbalanced porphyrin production and overall poorer survival in more aggressive cancers. These findings illuminate the anomalous porphyrin dynamics specifically within the tumor microenvironment, suggesting a potential target for therapeutic intervention.

FFAR-mediated signaling drives migration of pancreatic cancer cells in hypoxic fibroblast co-cultures

The tumor microenvironment (TME) comprises cancer and non-cancerous stromal cells, including fibroblasts. Free fatty acids (FFAs) regulate various biological responses by binding to G protein-coupled FFA receptors (FFARs). In this study, we examined the impact of FFAR1 and FFAR4 on the cell migration of pancreatic cancer PANC-1 cells co-cultured with 3T3 fibroblast cells under hypoxic conditions. PANC-1 cells cultured at 1 % O2 exhibited elevated FFAR1 expression and decreased FFAR4 expression compared to those at 21 % O2. Cell migration of PANC-1 cells was reduced under 1 % O2 conditions. FFAR1 knockdown enhanced PANC-1 cell migration, whereas FFAR4 knockdown inhibited it. Co-culture of PANC-1 cells with 3T3 cells at 1 % O2 significantly increased FFAR4 expression, while FFAR1 expression remained unchanged. To evaluate the effects of FFAR1 and FFAR4 on PANC-1 cell migration in co-culture with 3T3 cells, we conducted a wound healing assay using the Culture-Insert 2 Well. PANC-1 and 3T3 cells were individually seeded into the two wells and incubated at both 21 % and 1 % O2 for 13 h. The cell migration of PANC-1 cells co-cultured with 3T3 cells at 1 % O2 was notably higher compared to 21 % O2. TUG-770 reduced and TUG-891 enhanced the cell migration of PANC-1 cells co-cultured with 3T3 cells under both 21 % and 1 % O2 conditions. These findings suggest that FFAR1 and FFAR4 play important roles in regulating the cell migration of PANC-1 cells co-cultured with 3T3 cells under hypoxic conditions.

Glutathione Dynamics in the Tumor Microenvironment: A Potential Target of Cancer Stem Cells and T Cells.

Glutathione (GSH), the main cellular antioxidant, dynamically influences tumor growth, metastasis, and resistance to therapy in the tumor microenvironment (TME), which comprises cancer cells, immune cells, stromal cells, and non-cellular components, including the extracellular matrix, metabolites, hypoxia, and acidity. Cancer stem cells (CSCs) and T cells are minor but significant cell subsets of the TME. GSH dynamics influences the fate of CSCs and T cells. Here, we explored GSH dynamics in CSCs and T cells within the TME, as well as therapeutic approaches that could target these dynamics.

Diagnostic and Prognostic Markers for Pancreatitis and Pancreatic Ductal Adenocarcinoma.

Diagnostic markers are desperately needed for the early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity and the differential expression of PDA markers were identified by screening single cell (sc) RNAseq from tumor samples of a mouse model for PDA (KIC) at early and late stages of PDA progression compared to that of a normal pancreas. Candidate genes were identified from three sources: (1) an unsupervised screening of the genes preferentially expressed in mouse PDA tumors; (2) signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes, or genes encoding proteins that were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines; and (3) genes whose expression is associated with poor or better prognoses (PAAD, oncolnc.org). The developmental progression of PDA was detected in the temporal order of gene expression in the cancer cells of the KIC mice. The earliest diagnostic markers were expressed in epithelial cancer cells in early-stage, but not late-stage, PDA tumors. Other early markers were expressed in the epithelium of both early- and late-state PDA tumors. Markers that were expressed somewhat later were first elevated in the epithelial cancer cells of the late-stage tumors, then in both epithelial and mesenchymal cells, or only in mesenchymal cells. Stromal markers were differentially expressed in early- and/or late-stage PDA neoplasia in fibroblast and hematopoietic cells (lymphocytes and/or macrophages) or broadly expressed in cancer and many stromal cell types. Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and the acinar-specific homozygous deletion of differentiation transcription factors (dTFs), were screened for the early expression of all PDA markers identified in the KIC neoplasia. Prognostic markers associated with a more rapid decline were identified and showed differential and cell-type-specific expression in PDA, predominately in late-stage epithelial and/or mesenchymal cancer cells. Select markers were validated by immunohistochemistry in mouse and human samples of a normal pancreas and those with early- and late-stage PDA. In total, we present 2165 individual diagnostic and prognostic markers for disease progression to be tested in humans from pancreatitis to late-stage PDA.

Inferring single-cell spatial gene expression with tissue morphology via explainable deep learning.

The spatial arrangement of cells is vital in developmental processes and organogenesis in multicellular life forms. Deep learning models trained with spatial omics data uncover complex patterns and relationships among cells, genes, and proteins in a high-dimensional space, providing new insights into biological processes and diseases. State-of-the-art in silico spatial multi-cell gene expression methods using histological images of tissue stained with hematoxylin and eosin (H&E) to characterize cellular heterogeneity. These computational techniques offer the advantage of analyzing vast amounts of spatial data in a scalable and automated manner, thereby accelerating scientific discovery and enabling more precise medical diagnostics and treatments. In this work, we developed a vision transformer (ViT) framework to map histological signatures to spatial single-cell transcriptomic signatures, named SPiRiT ( S patial Omics P rediction and R eproducibility integrated T ransformer). Our framework was enhanced by integrating cross validation with model interpretation during hyper-parameter tuning. SPiRiT predicts single-cell spatial gene expression using the matched histopathological image tiles of human breast cancer and whole mouse pup, evaluated by Xenium (10x Genomics) datasets. Furthermore, ViT model interpretation reveals the high-resolution, high attention area (HAR) that the ViT model uses to predict the gene expression, including marker genes for invasive cancer cells ( FASN ), stromal cells ( POSTN ), and lymphocytes ( IL7R ). In an apple-to-apple comparison with the ST-Net Convolutional Neural Network algorithm, SPiRiT improved predictive accuracy by 40% using human breast cancer Visium (10x Genomics) dataset. Cancer biomarker gene prediction and expression level are highly consistent with the tumor region annotation. In summary, our work highlights the feasibility to infer spatial single-cell gene expression using tissue morphology in multiple-species, i.e., human and mouse, and multi-organs, i.e., mouse whole body morphology. Importantly, incorporating model interpretation and vision transformer is expected to serve as a general-purpose framework for spatial transcriptomics.

Complement or insult: the emerging link between complement cascade deficiencies and pathology of myeloid malignancies.

The complement cascade is an ancient and highly conserved arm of the immune system. The accumulating evidence highlights elevated activity of the complement cascade in cancer microenvironment and emphasizes its effects on the immune, cancer, and cancer stroma cells, pointing to a role in inflammation-mediated etiology of neoplasms. The role the cascade plays in development, progression, and relapse of solid tumors is increasingly recognized, however its role in hematological malignancies, especially those of myeloid origin, has not been thoroughly assessed and remains obscure. As the role of inflammation and autoimmunity in development of myeloid malignancies is becoming recognized, in this review we focus on summarizing the links that have been identified so far for complement cascade involvement in the pathobiology of myeloid malignancies. Complement deficiencies are primary immunodeficiencies that cause an array of clinical outcomes including an increased risk of a range of infectious as well as local or systemic inflammatory and thrombotic conditions. Here, we discuss the impact that deficiencies in complement cascade initiators, mid- and terminal-components and inhibitors have on the biology of myeloid neoplasms. The emergent conclusions indicate that the links between complement cascade, inflammatory signaling, and the homeostasis of hematopoietic system exist, and efforts should continue to detail the mechanistic involvement of complement cascade in the development and progression of myeloid cancers.

Elimination of radiation-induced senescent cancer cells and stromal cells invitro by near-infrared photoimmunotherapy.

Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as anovel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. NIR-PIT eliminates both senescent cancer and stromal cells invitro suggesting it may be a novel strategy for tumor treatment.

Cancer–stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

Abstract PO3-13-05: PD-L1 and PD-L2 Protein Expression is Frequently Discordant in Breast Cancer

Abstract Background: In neoadjuvant setting, PD-1 inhibitors have shown significantly higher pathological responses in patients (pts) with early-stage triple-negative breast cancer (TNBC) irrespective of PD-L1 status. However, durable responses are less common in estrogen receptor-positive (ER+) pts. Most breast cancer (BC) clinical trials of PD-1 inhibitors have thus far focused on PD-L1 expression for patient eligibility. The alternative PD-1 ligand, PD-L2, with reported ~4-fold greater affinity for PD-1, has been largely understudied. We recently reported that high cancer cell protein levels of PD-L2 in pts with treatment-naïve ER+ BC was an independent predictor of shorter progression-free survival. PD-L2 expression was significantly high in ER- group as well, however, given the low numbers of ER- pts, the study was not powered enough to determine the correlation of PD-L2 with PFS in the ER- subgroup. These findings suggest that PD-L2 has an important role in BC and combined PD-L1/PD-L2 status may help improve BC pt selection for PD-1 inhibitors. We therefore initiated efforts to determine baseline expression patterns of PD-L1 and PD-L2 in BC. Methods: PD-L1 and PD-L2 protein levels in cancer cells and tumor-infiltrating immune cells were prospectively analyzed by immunohistochemistry (IHC) using validated antibodies in diagnostic core biopsies of 31 consecutive pts diagnosed with localized or locoregional ER+/HER2- BC or TNBC. Percent positivity of PD-L1 and PD-L2 in cancer cells and immune cells was determined by our breast pathologist. Detectable PD-L1 or PD-L2 expression in ≥1% of cancer cells or stromal immune cells was considered positive. Spearman correlation and Wilcoxon paired analyses were used to measure and correlate PD-L1 and PD-L2 protein expression in cancer and immune cells. Results: There was no significant correlation between PD-L1 and PD-L2 expression, neither across all cases (N=31), nor within ER+ (N=22) or TNBC (N=9) cases. However, PD-L1 and PD-L2 expression patterns in BC differed in several ways. PD-L1-positivity in immune cells was higher than in cancer cells (median=5.0% vs. 0.0%; p=0.001), whereas PD-L2-positivity was higher in cancer cells than in immune cells (median=30% vs. 5.0%; p&amp;lt; 0.001). PD-L1 positivity in cancer cells and immune cells were positively correlated in TNBC (rho=0.69, p=0.04) but not in ER+ BC. Conversely, PD-L2 positivity in cancer cells and immune cells were positively correlated in ER+ BC (rho=0.68, p=0.001) but not in TNBC. TNBC diverged from ER+ BC by displaying higher PD-L1 positivity in immune cells (median=20.0% vs. 1.0%; p=0.004). By the conventional cut point for positivity of ≥1% for PD-L1 and PD-L2 in cancer cells or immune cells, all TNBC tumors were PD-L1-positive (9/9), with 8 also being PD-L2-positive. Of the 22 ER+ cases, 16 were PD-L2-positive, of which only 9 were also PD-L1-positive. Table 1. shows cross tabulation data for PD-L1 and PD-L2 status by BC subtype. Conclusions: PD-L1 and PD-L2 proteins show divergent expression and are not correlated in BC. Discordant PD-L2 and PD-L1 expression may be more common in ER+ BC than in TNBC. This finding justifies efforts to explore PD-L2 as a complementary marker to PD-L1 for improved prediction of response to PD-1 inhibitors, which may benefit patients with aggressive ER+ BC that are eligible for chemotherapy. Table 1. PD-L1 vs. PD-L2 Status by Breast Cancer Subtype Citation Format: Lubna Chaudhary, Julie Jorns, Yunguang Sun, Sailaja Kamaraju, Yee Chung Cheng, Amanda Kong, Tina Yen, Caitlin Patten, Chandler Cortina, Inna Chervoneva, Christopher Chitambar, Hallgeir Rui. PD-L1 and PD-L2 Protein Expression is Frequently Discordant in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-05.

Abstract PO4-26-03: Collagen deposition in the breast cancer microenvironment correlates with ER-positive, Histological Grade I and low proliferation tumors

Abstract Breast cancer is a highly heterogeneous neoplasm both at the molecular and histological levels. Breast cancer cells present extremely high levels of genetic heterogeneity concerning mutational burden, copy number variations (CNVs) and epigenetic signatures. While this level of heterogeneity is well established, other layers of heterogeneity, such as tumor microenvironment (TME) heterogeneity, remains to be elucidated. TME in breast cancer is an intricate network encompassing tumor cells, “normal” stroma cells such as fibroblasts, lymphocytes, and a plethora of extracellular matrix (ECM) proteins produced either by cancer cells or stromal cells. These three interacting modules (cancer cells, stromal cells and the ECM) can affect each other in a reciprocal way with profound effects on patient prognosis and treatment outcome. Major protein components of the TME are ECM-related collagens, that are produced by breast cancer cells or/and cancer associated fibroblasts. The ECM collagen in breast cancer, that is mainly composed of collagen types I, III and V, has multiple roles modulating tumor growth, cancer cell invasiveness and metastasis. Here by Masson Trichrome and Immunohistochemistry/Aniline Blue co-stains for collagen fibers and proliferation index (Ki-67) in breast cancer FFPE tissue samples, we identified a statistically significant correlation between collagen deposition and Estrogen Receptor (ER)) status (p&amp;lt; 0.05, proliferation index (p&amp;lt; 0.01) and histological Grade (p=0.01). Further, in order to expand and support our findings, we analyzed the METABRIC study through the cBioPortal hub in relevance to collagen genes and multiple clinicopathological parameters. We were able to identify a coregulated subset of collagen genes that presented a positive correlation with ER-positive (p&amp;lt; 10−10, q&amp;lt; 10−10), low proliferating (p&amp;lt; 10−10, q&amp;lt; 10−10), Grade I (p=2.35e−9, q&amp;lt; 1.07e−8) group of breast cancer cases, supporting our findings. In addition, analyses of collagen-high patients from the METABRIC study indicated that most cases presented with PIK3CA, CBFB and MAP3K1 mutations, while through the g.Profiler software we identified collagen-high relevant gene signatures that encompassed gene ontology terms related to tissue development, extracellular matrix organization and cell migration. Collectively, our preliminary results indicate that ECM collagen deposition in breast cancer is characteristic of low-grade tumors with a better differentiation and possibly with a favorable prognosis. Citation Format: Angelos Koutras, Chaido Sirinian, Olga Bogri, Martha Nifora, Maria Theakou, Soren Degn, Ioannis Mouratidis, Dimitrios Chaniotis, Stavros Peroukidis, Ilias Georgakopoulos-Soares, Anastasios Papanastasiou. Collagen deposition in the breast cancer microenvironment correlates with ER-positive, Histological Grade I and low proliferation tumors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-03.

Abstract LB190: Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer

Abstract In the context of the tumor microenvironment (TME), a substantial increased secreted protein, chitinase-3 like-protein-1 (CHI3L1) has been observed, emanating from various cell types such as cancer cells, macrophages, and T cells. The development of an immunosuppressive and fibrotic TME has been implicated in CHI3L1-mediated cancer progression. Despite recognizing the potential for CHI3L1 blockade to facilitate cancer regression, there remains a limited understanding of the regulatory mechanisms governing the expression of this tumor-promoting protein within cancer cells. Our investigations, along with others, have identified low levels of CHI3L1 protein in normal epithelial cells, in stark contrast to its abundant presence in pancreatic cancer tumor specimens. We propose that the interaction between cancer and stromal cells in the TME induces CHI3L1 expression within cancer cells. To test this hypothesis, we established a co-culture system involving cancer and stromal cells including pancreatic stellate cells, macrophages and T cells to emulate the TME of pancreatic cancer. Subsequently, we observed a ten-fold increase in CHI3L1 expression in cancer cells upon exposure to co-culture media. Through secretomic analysis, C-X-C motif chemokine ligand 10 (CXCL10) emerged as a potential inducer of CHI3L1 expression in response to co-culture media. CXCL10/CXCR3 signaling has been reported to correlate with poor prognosis in pancreatic cancer, while their role in disease development is still unclear. Concurrently, Gene Set Enrichment Analysis (GSEA) identified the activation of TNFα signaling via NF-κB and TGF-β pathways in pancreatic cancer patients with high CHI3L1 expression. Notably, chromatin-immunoprecipitation and promoter activity assays validated the direct binding and transcriptional regulatory role of NF-κB, a downstream transcription factor of CXCL10, on CHI3L1 gene in cancer cells. In addition, epigenetic reprogramming occurred during co-culture media education. Our observations indicated demethylation of the CHI3L1 promoter during co-culture media exposure, a phenomenon confirmed to be mediated by STAT3 activation. In summary, this study elucidates, for the first time, the intricate crosstalk between cancer and stromal cells within the TME of pancreatic cancer as a pivotal factor for the transcriptional and epigenetic dysregulation of CHI3L1, which then contributes to TME remodeling and malignant pancreatic cancer progression. Citation Format: Pei-Chia (Peggy) Su, Yi-Ching Wang. Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB190.

Gastric Cancer With the Increased Nicotinamide N-methyltransferase-positive Stromal Cells Includes Unfavorable Prognosis-related Cancer-associated Fibroblasts.

"Stromal high expression" of Nicotinamide N-methyltransferase (NNMT), previously reported as a poor prognostic factor of gastric cancer, was based on immunohistochemical H-score. However, this could simply indicate an increase in cancer-associated fibroblasts (CAFs) because NNMT is positive for fibroblasts. To verify this, the proportion and staining intensity of stromal NNMT-positive stellate/spindle cells were evaluated separately and examined for its association with related proteins (H3K4me3, H3K27me3, and LOXL2). Immunohistochemistry for NNMT, H3K4me3, H3K27me3, and LOXL2 was performed on 521 tissue microarrays of gastric cancer. Cancer stromal stellate/spindle cells were evaluated according to morphology, proportion, and stain intensity of NNMT, loss of H3K4me3 and H3K27me3, and stain intensity of LOXL2. Their associations with clinicopathological characteristics and overall survival were examined. Higher staining intensity of NNMT was not related to a poorer prognosis. However, higher proportion of NNMT-positive stellate/spindle cells indirectly contributed to a poor prognosis. It was associated with CAF-like morphology and a global decrease in H3K4me3/H3K27me3, which were both associated with high LOXL2 expression. These three factors were independent poor prognostic factors. In addition, in the LOXL2-high group, prognosis significantly deteriorated with the presence of a global decrease in H3K4me3/H3K27me3. The higher proportion of NNMT-positive cancer stromal cells in gastric cancer serves as an indicator for identifying unfavorable prognostic CAFs that show a global decrease in H3K4me3/H3K27me3. This facilitates research on the nature of these cells and their characteristics.

Abstract 2830: Intravital multiphoton microscopy of bone metastatic renal cell carcinoma

Abstract Clear cell renal carcinoma (ccRCC) represents 80% of renal neoplasms, with bone as a major site for distant spread (35-40% of patients). These secondary lesions cause a variety of skeletal-related complications, including pain, spinal cord compression, hypercalcemia, mobility issues and fractures, posing a significant negative impact on patient quality of life and survival. Furthermore, bone is well-known as a site of therapy resistance, which confers to patients treated with systemic therapies significantly worse time-to-treatment failure and progression-free and overall survival compared to those without bone metastases. Because of the complexity of the bone microenvironment and the paucity of suitable models, preclinical investigation of bone metastasis and therapy response is challenging. Preclinical models of bone metastasis that integrate tissue engineering and advanced imaging techniques can facilitate the study of the mechanism of tumor progression and therapy response. We recently developed a window model amenable to intravital multiphoton microscopy (MPM) to longitudinally monitor human ccRCC lesions in tissue engineered bone constructs (TEBCs) in immunodeficient mice. TEBCs were generated by functionalizing polymeric polycaprolactone scaffolds with bone morphogenetic protein 7. Human ccRCC lesions, after implantation into the bone cavity, were longitudinally monitored for growth and niche development, using multi-parameter recording of collagen/bone matrix (SHG), bone surface (THG), blood vessels/stromal phagocytes (fluorescent dextran), and tumor cells (GFP). However, this model lacked key components of the immune system (including B and T-cells), which are of major interest in the study of tumor progression and (immune) therapy response. Therefore, we developed a tissue-engineered preclinical model that incorporate species-specific interactions between cancer and bone stromal cells, and account for an intact immune system. To this purpose, we optimized TEBC formation in C57BL/6 fluorescent reporter mice (Sp7-mCherry, osteoblasts; Trap-td-Tomato, osteoclasts; GFP, immune cells, αSMA-RFP fibroblasts; and Flk1-GFP, blood vessels), coupled to detection by intravital or ex vivo multiphoton microscopy analysis, to monitor the evolution and fate of the TEBC. Interestingly, the bone marrow in the core of the TEBC was replaced by a desmoplastic tissue with features like the foreign body response, including foreign body giant cells, which was not present in immunodeficient mice. By applying dual color GFP/αSMA-RFP mice, which expressed red fluorescence in activated fibroblasts, we confirmed the fibrotic nature of this core. To avoid its formation, we generated scaffold-free ossicle, by direct implantation of BMP7 and VEGF in the subcutaneous tissue. To conclude, we established a protocol for generating ectopic ossicles in immunocompetent mice. Citation Format: Stefan Maksimovic, Nina Constanza Boscolo, Sergio Barrios, Antonios Mikos, Matthew T. Campbell, Eleonora Dondossola. Intravital multiphoton microscopy of bone metastatic renal cell carcinoma [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 2830.

Abstract 3645: Unraveling spatial complexity of the tumor microenvironment: A whole transcriptomic perspective with Visium HD

Abstract In recent years, advances in spatial transcriptomics have revolutionized our understanding of the tumor microenvironment, providing crucial insights into the complex interplay of different cell types within cancer tissues. In this study, we employed the new, high definition Visium spatial transcriptomics assay (Visium HD) to investigate the intricate molecular landscape of prostate cancer at single-cell scale resolution and across the whole transcriptome. Our research focused on deciphering the spatial heterogeneity of gene expression patterns within the tumor microenvironment, shedding light on the interactions between cancer cells, stromal cells, and vasculature. The Visium HD assay enables an unbiased exploration of the transcriptome on FFPE tissue sections mounted on standard glass slides. Combining gene expression data with H&amp;E images from the same section, it allows precise characterization of local molecular features and disease states at single-cell scale. We analyzed a set of human prostate adenocarcinoma samples with the Visium HD assay, and identified spatially regulated gene signatures associated with specific cell types and functional pathways. Our findings have recapitulated previously identified molecular markers, including VEGFA and MYC which have been implicated in tumor progression and angiogenesis. The spatial gene expression patterns had good correlation with histological annotations of cancerous and normal tissue within the H&amp;E image. However, Visium HD allowed us to further observe molecular processes and pathways specific to this adenocarcinoma sample. This integrative approach offers valuable implications for personalized cancer therapy and the development of targeted interventions tailored to the spatial context of individual tumors. Our study underscores the significance of understanding the spatial organization of the whole transcriptome in cancer tissues, and highlights the potential of the Visium HD platform as a powerful tool for unraveling the complexities of the tumor microenvironment. These insights pave the way for the development of innovative therapeutic strategies and precision medicine approaches, ultimately contributing to improved outcomes for cancer patients. Citation Format: Yifeng Yin, Jerald Sapida, David Sukovich, David Patterson, Augusto Tentori. Unraveling spatial complexity of the tumor microenvironment: A whole transcriptomic perspective with Visium HD [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 3645.