Co-culture Of Cancer Cells Research Articles

Functional gene signature offers a powerful tool for characterizing clinicopathological features and depicting tumor immune microenvironment of colorectal cancer

BackgroundColorectal cancer, a prevalent malignancy worldwide, poses a significant challenge due to the lack of effective prognostic tools. In this study, we aimed to develop a functional gene signature to stratify colorectal cancer patients into different groups with distinct characteristics, which will greatly facilitate disease prediction.ResultsPatients were stratified into high- and low-risk groups using a prediction model built based on the functional gene signature. This innovative approach not only predicts clinicopathological features but also reveals tumor immune microenvironment types and responses to immunotherapy. The study reveals that patients in the high-risk group exhibit poorer pathological features, including invasion depth, lymph node metastasis, and distant metastasis, as well as unfavorable survival outcomes in terms of overall survival and disease-free survival. The underlying mechanisms for these observations are attributed to upregulated tumor-related signaling pathways, increased infiltration of pro-tumor immune cells, decreased infiltration of anti-tumor immune cells, and a lower tumor mutation burden. Consequently, patients in the high-risk group exhibit a diminished response to immunotherapy. Furthermore, the high-risk group demonstrates enrichment in extracellular matrix-related functions and significant infiltration of cancer-associated fibroblasts (CAFs). Single-cell transcriptional data analysis identifies CAFs as the primary cellular type expressing hub genes, namely ACTA2, TPM2, MYL9, and TAGLN. This finding is further validated through multiple approaches, including multiplex immunohistochemistry (mIHC), polymerase chain reaction (PCR), and western blot analysis. Notably, TPM2 emerges as a potential biomarker for identifying CAFs in colorectal cancer, distinguishing them from both colorectal cancer cell lines and normal colon epithelial cell lines. Co-culture of CAFs and colorectal cancer cells revealed that CAFs could enhance the tumorigenic biofunctions of cancer cells indirectly, which could be partially inhibited by knocking down CAF original TPM2 expression.ConclusionsThis study introduces a functional gene signature that effectively and reliably predicts clinicopathological features and the tumor immune microenvironment in colorectal cancer. Moreover, the identification of TPM2 as a potential biomarker for CAFs holds promising implications for future research and clinical applications in the field of colorectal cancer.

Enhancing cancer immunotherapy using cordycepin and Cordyceps militaris extract to sensitize cancer cells and modulate immune responses

Integrating immunotherapy with natural compounds holds promise in enhancing the immune system’s ability to eliminate cancer cells. Cordyceps militaris, a traditional Chinese medicine, emerges as a promising candidate in this regard. This study investigates the effects of cordycepin and C. militaris ethanolic extract (Cm-EE) on sensitizing cancer cells and regulating immune responses against breast cancer (BC) and hepatocellular carcinoma (HCC) cells. Cordycepin, pentostatin and adenosine were identified in Cm-EE. Cordycepin treatment decreased HLA-ABC-positive cells in pre-treated cancer cells, while Cm-EE increased NKG2D ligand and death receptor expression. Additionally, cordycepin enhanced NKG2D receptor and death ligand expression on CD3-negative effector immune cells, particularly on natural killer (NK) cells, while Cm-EE pre-treatment stimulated IL-2, IL-6, and IL-10 production. Co-culturing cancer cells with effector immune cells during cordycepin or Cm-EE incubation resulted in elevated cancer cell death. These findings highlight the potential of cordycepin and Cm-EE in improving the efficacy of cancer immunotherapy for BC and HCC.

Abstract A036: The role of hypoxia in CD8+ T cell localization and function in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to existing therapeutic options. This resistance to treatment is partly due to its extensive inflammatory, desmoplastic stromal response, along with a hypoxic microenvironment. Although tumor hypoxia induces adaptive responses in both cancer cells and the surrounding stroma, most studies on the role of hypoxia in tumorigenesis have focused on cancer cell-intrinsic properties. The impact of hypoxia on stromal cells themselves and their interactions with cancer cells has remained largely unknown. We previously showed that inflammatory cancer-associated fibroblasts (iCAFs), which express high levels of cytokines and chemokines, are preferentially located in hypoxic regions of PDAC, and that hypoxia promotes an inflammatory fibroblast phenotype. The iCAF phenotype has been correlated with highly immunosuppressive features of PDAC. These findings and our preliminary observation that CD8+ T cells are largely excluded from hypoxic PDAC regions, raise the possibility that hypoxia suppresses the infiltration and activation of CD8+ T cells by modulating their interactions with inflammatory fibroblasts. By culturing CD8+ T cells under hypoxia with conditioned media from co-cultures of pancreatic cancer cells and fibroblasts, we have found that cancer cells and fibroblasts decrease CD8+ T cell proliferation and cytotoxic activity under low oxygen conditions. We are working to elucidate the mechanism by which hypoxia regulates CD8+ T cell infiltration and activation through cancer cell-fibroblast crosstalk. Citation Format: Ashley M. Mello, Tenzin Ngodup, Katelyn L. Donahue, Marina Pasca Di Magliano, Kyoung Eun Lee. The role of hypoxia in CD8+ T cell localization and function in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A036.

Targeting the cancer cells and cancer-associated fibroblasts with next-generation FGFR inhibitors in prostate cancer co-culture models.

Inhibition of androgen receptor (AR) signaling is the main treatment strategy in advanced prostate cancer (PCa). A subset of castration resistant prostate cancer (CRPC) bypasses the AR blockade by increased fibroblast growth factor receptor (FGFR) signaling. The first- and second-generation, non-covalent FGFR inhibitors (FGFRis) have largely failed in the clinical trials against PCa. In this study, we tested the drug sensitivity of LNCaP, VCaP, and CWR-R1PCa cell lines to second-generation, covalent FGFRis (FIIN1, FIIN2) and a novel FGFR downstream molecule inhibitor (FRS2αi). 2D and 3D mono- and co-cultures of cancer cells, and cancer-associated fibroblasts (CAFs) were used to mimic tumor-stroma interactions in the extracellular matrix (ECM). The treatment responses of the FGFR signaling molecules, the viability and proliferation of cancer cells, and CAFs were determined through immunoblotting, migration assay, cell viability assay, and real-time imaging. Immunofluorescent and confocal microscopy images of control and treated cultures of cancer cells and CAFs, and their morphometric data were deduced. The FGFRis were more effective in mono-cultures of the cancer cells compared with co-cultures with CAFs. The FRS2αi was specifically effective in co-cultures with CAFs but was not cytotoxic to CAF mono-cultures as in the case of FIIN1 and FIIN2. At the molecular level, FRS2αi decreased p-FRS2α, p-ERK1/2, and activated apoptosis as monitored by cleaved caspase-3 activity in a concentration-dependent manner in the co-cultures. We observed no synergistic drug efficacy in the combination treatment of the FGFRi with ARi, enzalutamide, and darolutamide. The FRS2αi treatment led to a decrease in proliferation of cancer cell clusters in co-cultures as indicated by their reduced size and Ki67 expression. CAFs exert a protective effect on cancer cells and should be included in the invitro models to make them physiologically more relevant in screening and testing of FGFRis. The FRS2αi was the most potent agent in reducing the viability and proliferation of the 3D organotypic co-cultures, mainly by disrupting the contact between CAFs and cancer cell clusters. The next-generation FGFRi, FRS2αi, may be a better alternative treatment option for overcoming ARi treatment resistance in advanced PCa.

A phosphoglycerate mutase 1 allosteric inhibitor restrains TAM-mediated colon cancer progression

Colorectal cancer (CRC) is a prevalent malignant tumor often leading to liver metastasis and mortality. Despite some success with PD-1/PD-L1 immunotherapy, the response rate for colon cancer patients remains relatively low. This is closely related to the immunosuppressive tumor microenvironment mediated by tumor-associated macrophages (TAMs). Our previous work identified that a phosphoglycerate mutase 1 (PGAM1) allosteric inhibitor, HKB99, exerts a range of anti-tumor activities in lung cancer. Here, we found that upregulation of PGAM1 correlates with increased levels of M2-like tumor-associated macrophages (TAMs) in human colon cancer samples, particularly in liver metastatic tissues. HKB99 suppressed tumor growth and metastasis in cell culture and syngeneic tumor models. M2-polarization, induced by colon cancer cell co-culture, was reversed by HKB99. Conversely, the increased migration of colon cancer cells by M2-TAMs was remarkably restrained by HKB99. Notably, a decrease in TAM infiltration was required for the HKB99-mediated anti-tumor effect, along with an increase in CD8+ T cell infiltration. Moreover, HKB99 improved the efficacy of anti-PD-1 treatment in syngeneic tumors. Overall, this study highlights HKB99's inhibitory activity in TAM-mediated colon cancer progression. Targeting PGAM1 could lead to novel therapeutic strategies and enhance the effectiveness of existing immunotherapies for colon cancer.

TUMOR-INFILTRATING NOCICEPTOR NEURONS PROMOTE IMMUNOSUPPRESSION.

Nociceptor neurons impact tumor immunity. Removing nociceptor neurons reduced myeloid-derived suppressor cell (MDSCs) tumor infiltration in mouse models of head and neck carcinoma and melanoma. Carcinoma-released small extracellular vesicles (sEVs) attract nociceptive nerves to tumors. sEV-deficient tumors fail to develop in mice lacking nociceptor neurons. Exposure of dorsal root ganglia (DRG) neurons to cancer sEVs elevated expression of Substance P, IL-6 and injury-related neuronal markers while treatment with cancer sEVs and cytotoxic CD8 T-cells induced an immunosuppressive state (increased exhaustion ligands and cytokines). Cancer patient sEVs enhanced DRG responses to capsaicin, indicating increased nociceptor sensitivity. Conditioned media from DRG and cancer cell co-cultures promoted expression of MDSC markers in primary bone marrow cells while DRG conditioned media together with cancer sEVs induced checkpoint expression on T-cells. Our findings indicate that nociceptor neurons facilitate CD8+ T cell exhaustion and enhance MDSC infiltration. Targeting nociceptor-released IL-6 emerges as a novel strategy to disrupt harmful neuro-immune interactions in cancer and enhance anti-tumor immunity.

Selective anticancer effects of plasma-activated saline in 3D tumor model co-culturing of normal cells and cancer cells

Compared with conventional two-dimensional (2D) cell culture model, the 3D tumor model constructed in vitro is better representative of the tumor microenvironment in vivo. Here, we proposed the utilization of 3D tumor model of co-cultured cancer cells and normal cells to evaluate the selective anticancer effects of cold atmospheric plasma-activated saline (PAS), and expected to provide more precise information about PAS-tumor interactions. By cell sorting, we clarified that A375 melanoma cells and HaCaT normal skin cells purified from the 3D multicellular tumor model differ in sensitivity and responsiveness to PAS compared to the 2D culture model. And during the optimization of PAS treatment parameters, we further found that A375 cells were almost completely killed while HaCaT cells were still present in large numbers after 5 d of certain PAS treatment. Our experiment innovatively carries out the selective study of plasma technology in 3D co-culture system and provides a theoretical basis for further clinical and practical applications of PAS.

Reciprocal Interactions of Human Monocytes and Cancer Cells in Co-Cultures In Vitro.

The tumor microenvironment (TME) includes immune and stromal cells and noncellular extracellular matrix (ECM) components. Tumor-associated macrophages (TAMs) are the most important immune cells in TME and are crucial for carcinomas' progression. The purpose was to analyze direct and indirect interactions in co-culture of tumor cells with monocytes/macrophages and, additionally, to indicate which interactions are more important for cancer development. Cytokines, reactive oxygen species, nitric oxide level, tumor cell cycle and changes in tumor cell morphology after human tumor cells (Hep-2 and RK33 cell lines) with human monocyte/macrophage (THP-1 cell line) interactions were tested. Morphology and cytoskeleton organization of tumor cells did not change after co-culture with macrophages. In co-culture of tumor cells with human monocyte, changes in the percentage of tumor cells in cell cycle phases was observed. No significant changes in reactive oxygen species (ROS) were found in the co-culture as compared to the tumor cell mono-culture. Monocytes produced about three times higher ROS than tumor cells. In co-cultures, a lower nitric oxide (NOx) level was found as compared to the sum of the production by both mono-cultures. Co-culture conditions limited the production of cytokines (IL-4, IL-10 and IL-13) as compared to the sum of their level in mono-cultures. In conclusion, macrophages influence tumor cell growth and functions. Mutual (direct and paracrine) interactions between tumor cells and macrophages changed cytokine production and tumor cell cycle profile. The data obtained may allow us to initially indicate which kind of interactions may have a greater impact on cancer development processes.

Evaluation of Tumorigenic Properties of MDA-MB-231 Cancer Stem Cells Cocultured with Telocytes and Telocyte-Derived Mitochondria Following miR-146a Inhibition.

Telocytes have some cytoplasmic extensions called telopodes, which are thought to play a role in mitochondrial transfer in intercellular communication. Besides, it is hypothesized that telocytes establish cell membrane-mediated connections with breast cancer cells in coculture and may contribute to the survival of neoplastic cell clusters together with other stromal cells. The aim of this study is to investigate the contribution of telocytes and telocyte-derived mitochondria, which have also been identified in breast tumors, to the tumor development of breast cancer stem cells (CSCs) via miR-146a-5p. The isolation/characterization of telocytes from bone marrow mononuclear cells and the isolation of mitochondria from these cells were performed, respectively. In the next step, CSCs were isolated from the MDA-MB-231 cell line and were characterized. Then, miR-146a-5p expressions of CSCs were inhibited by anti-miR-146a-5p. The epithelial-mesenchymal transition (EMT) was determined by evaluating changes in vimentin protein levels and was evaluated by analyzing BRCA1, P53, SOX2, E-cadherin, and N-cadherin gene expression changes. Our results showed that miR-146a promoted stemness and oncogenic properties in CSCs. EMT (N-cadherin, vimentin, E-cadherin) and tumorigenic markers (BRCA1, P53, SOX2) of CSCs decreased after miR-146a inhibition. Bone marrow-derived telocytes and mitochondria derived from telocytes favored the reduction of CSC aggressiveness following this inhibition.

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.

A2AR-mediated CXCL5 upregulation on macrophages promotes NSCLC progression via NETosis

Tumor-associated macrophages (TAMs) are abundant in tumors and interact with tumor cells, leading to the formation of an immunosuppressive microenvironment and tumor progression. Although many studies have explored the mechanisms underlying TAM polarization and its immunosuppressive functions, understanding of its progression remains limited. TAMs promote tumor progression by secreting cytokines, which subsequently recruit immunosuppressive cells to suppress the antitumor immunity. In this study, we established an in vitro model of macrophage and non-small cell lung cancer (NSCLC) cell co-culture to explore the mechanisms of cell-cell crosstalk. We observed that in NSCLC, the C-X-C motif chemokine ligand 5 (CXCL5) was upregulated in macrophages because of the stimulation of A2AR by adenosine. Adenosine was catalyzed by CD39 and CD73 in macrophages and tumor cells, respectively. Nuclear factor kappa B (NFκB) mediated the A2AR stimulation of CXCL5 upregulation in macrophages. Additionally, CXCL5 stimulated NETosis in neutrophils. Neutrophil extracellular traps (NETs)-treated CD8+ T cells exhibited upregulation of exhaustion-related and cytosolic DNA sensing pathways and downregulation of effector-related genes. However, A2AR inhibition significantly downregulated CXCL5 expression and reduced neutrophil infiltration, consequently alleviating CD8+ T cell dysfunction. Our findings suggest a complex interaction between tumor and immune cells and its potential as therapeutic target.

Induction of necroptosis in multinucleated giant cells induced by conditionally replicating syncytial oHSV in co-cultures of cancer cells and non-cancerous cells

Viral modifications enabling syncytium formation in infected cells can augment lysis by oncolytic herpes simplex viruses (oHSVs) which selectively kill cancer cells. In the case of receptor-retargeted oHSV (RR-oHSV) that exclusively enter and spread to cancer cells, anti-tumor effects can be enhanced in a magnitude of >100,000-fold by modifying the virus to a syncytial type (RRsyn-oHSV). However, when syncytia containing non-cancerous cells are induced by conditionally-replicating syncytial oHSV (CRsyn-oHSV), syncytial death occurs at an early stage. This results in limited anti-tumor effects of the CRsyn-oHSV. Here, we investigated whether necroptosis is involved in death of the syncytia formed by the fusion of cancer cells and non-cancerous cells. Mixed-lineage kinase domain-like (MLKL), a molecule executing necroptosis, was expressed in all murine cancer cell lines examined, while receptor-interacting protein kinase 3 (RIPK3), which phosphorylates MLKL, was absent from most cell lines. In contrast, RIPK3 was expressed in non-cancerous murine fibroblast cell lines. When a CRsyn-oHSV-infected RIPK3-deficient cancer cell line was co-cultured with the fibroblast cell line, but not with the cancer cells themselves, MLKL was phosphorylated and syncytial death was induced. These results indicate that early necroptosis is induced in multinucleated giant cells formed by CRsyn-oHSV when they also contain non-cancerous cells.

Abstract LB352: Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant cancer

Abstract The utilization of messenger RNA (mRNA)-based cancer vaccines represents a compelling therapeutic strategy, providing distinct advantages over alternative modalities such as peptide vaccines or small-molecule-based cancer therapies. This innovative approach enables the concurrent targeting of heterogeneous cancer populations characterized by multiple mutations while preserving the integrity of normal cells. The Kristen rat sarcoma viral oncogene (KRAS) mutation is prevalent in lung, pancreatic, and colon cancer, and patients harboring KRAS mutations face a particularly dismal prognosis. This study focuses on the optimization of an mRNA-based cancer vaccine designed to target various KRAS mutations, demonstrating its efficacy inducing potent anti-tumor immune responses in both ex vivo immune cell and cancer cell co-culture, as well as in vivo syngeneic mouse cancer models. For the ex vivo evaluation of the multi-targeting capabilities of the vaccine, human peripheral blood mononuclear cells (PBMCs) primed with our mRNA cancer vaccine were subsequently co-incubated with human normal and cancer cell lines harboring KRAS mutations to assess antigen-specific immune responses and the normal cell-sparing effect of the cancer vaccine. Four types of KRAS mutant cancer cells co-cultured with the vaccine-activated PBMCs showed substantially decreased cell viability. We found enrichment of cytotoxic and pro-inflammatory cytokines including ganzyme B, IFNγ, IL-6 and IL-10 in co-culture system with KRAS vaccine transfected monocyte and PBMCs. This suggests that multiple KRAS mutations can be targeted with a single type of mRNA vaccine candidate. In the LL/2 syngeneic model (murine Lewis lung carcinoma, KRAS G12C mutant), the in vivo efficacy of KRAS cancer vaccine candidates was evaluated, revealing a notable reduction in tumor growth in response to the vaccine treatment. Also, during the treatment period, there were no significant clinical symptoms in the mouse between groups indicating the vaccine doses were tolerable. These results indicate that the KRAS vaccine effectively inhibits the growth of an LL/2 tumor which has the immunosuppressive tumor microenvironment. The ability to address the challenges posed by diverse KRAS mutations underscores the promising potential of this multiple-KRAS mutation targeting mRNA-based cancer vaccine in the pursuit of enhanced therapeutic outcomes for patients afflicted with KRAS-mutant cancer. Citation Format: Chang Gyu Lim, Seung-Hyun Shin, Youngjin Han, Yong Ho Heo, Innah Kim, Ji Hee Lee, Miyoung Lee, Seongju Jeong, Yunjae Kim, Jooyun Byun, Daejin Kim, In Young Choi. Unraveling therapeutic potentials of mRNA-based cancer vaccine concurrently targeting heterogeneous KRAS mutant 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 LB352.

Abstract LB317: TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance

Abstract Triple negative breast cancer (TNBC) carries a poorer prognosis than other breast cancer subtypes due to high metastatic incidence, lack of molecular drug targets, and rapid development of chemoresistance. Cancer-associated fibroblasts (CAFs) are key extrinsic drivers of chemoresistance in primary TNBC tumors, but it is unknown whether resident fibroblasts in distant metastatic tissues also influence TNBC drug sensitivity. Since the lung is a top metastatic site in TNBC, we developed a breast cancer cell (BCC) - primary human donor-derived lung fibroblast (LF) co-culture model to identify therapeutic vulnerabilities and mechanisms of fibroblast-mediated extrinsic chemoresistance in lung-metastatic TNBC. We characterized the growth and chemoresistance phenotypes of BCC co-cultured with primary LFs via bioluminescence and fluorescence imaging and evaluated their transcriptional and secretory profiles using bulk and scRNAseq and multiplex cytokine analysis. Co-culture of human BCC and primary LFs resulted in BCC molecular subtype-dependent changes in cell growth and drug resistance compared with conventional monoculture. Moreover, we also observed increased LF secretion of the immunomodulatory cytokines IL-6 and CCL2 in response to both BCC co-culture and BCC-conditioned media. Transcriptomic analysis of LFs sorted from co-culture identified upregulation of pro-inflammatory and pro-fibrotic iCAF and myCAF-like gene signatures, while co-cultured TNBC cells upregulated interferon response-mediated multi-therapy resistance genes and cellular dormancy signatures. We applied this model in a high throughput screen of 846 kinase inhibitor compounds, which identified reproducible differential BCC responses to kinase inhibitors between monoculture and co-culture conditions. When co-cultured with LFs, BCC were broadly more resistant to kinase inhibitor compounds but showed increased sensitivity to inhibitors of the lipid kinase VPS34, an important initiator of autophagy. We subsequently established that autophagy is increased in co-cultured TNBC cells and contributes to LF-mediated resistance to kinase inhibitors. Moreover, we determined that while paracrine signals from BCC can promote LF activation indicated by enhanced cytokine secretion, cell-cell contact is required for LF modulation of TNBC therapy responses in co-culture. Thus, we conclude that breast cancer cell co-culture with primary lung fibroblasts is a scalable in vitro model that can facilitate therapeutic discovery and mechanistic evaluation of extrinsic modulation of therapy responses in metastatic breast cancer, and further demonstrate that BCC can promote paracrine activation of co-cultured LFs in association with juxtacrine induction of BCC autophagy and extrinsic therapy resistance. Citation Format: Marika L. Klosowski, Kathryn Cronise, Claire Stratton, Qiong Zhou, Hector Esquer, Daniel LaBarbera, Daniel Regan. TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance [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 LB317.

Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Abstract 5529: Mimicking CAFs, pial cells support leptomeningeal metastasis

Abstract Leptomeningeal metastasis (LM), the spread of cancer into cerebrospinal fluid (CSF)-filled coverings of the brain and spinal cord, is a dismal complication of cancer leading to rapid neurologic disability and death. While a diverse array of primary tumors may result in LM, recent work from our laboratory (Chi Y et al 2020 Science) demonstrates a conserved transcriptional signature, independent of primary tumor, suggesting that tumor-microenvironmental interactions play a dominant role in this pathology. We have proteomically interrogated CSF collected from breast (n = 45), lung (n = 30), and melanoma (n = 27) patients with and without LM. We detect robust expression of inflammatory cytokines in all LM+ samples, 15 of these cytokines including IL8, IL6, and CXCL1 are conserved across tumor types in the presence of LM. Because cancer cells in the CSF do not express mRNA associated with these cytokines, we hypothesized that these inflammatory cytokines may originate from the microenvironment. Within the leptomeninges, LM cells adhere to and interact with the innermost layer of the meninges, the pia mater, which contains fibroblast-like cells. Co-culture of cancer cells with pial cells induced changes in the pial cell transcriptome and secretome, leading to increased IL8, IL6, and CXCL1 production. This communication between cancer cells and pial cells is preserved in the absence of direct cell-cell contact. These pial cells support cancer cell growth in vivo, as measured by co-implantation experiments. In addition, bulk RNA sequencing of mouse pial cells shows an inflammatory signature upregulated in the presence of LM. Taken together, these observations support a cancer-associated-fibroblast like role for pial cells in the setting of LM. Our work extends the CAF phenotype beyond classical fibroblasts to include pial cells, suggesting novel therapeutic opportunities within CNS malignancies. Citation Format: Jenna Snyder, Jan Remsik, Adrienne Boire. Mimicking CAFs, pial cells support leptomeningeal metastasis [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 5529.

Abstract 3935: Involvement of nanoscale physical communication in obesity-associated breast cancer severity

Abstract Obesity is a risk factor for several cancers, including breast cancer, and contributes up to 20% of cancer-related deaths. Growing evidence from both clinical and preclinical studies indicates that increased obesity is associated with increases in cancer incidence, progression, metastasis, and therapeutic resistance. According to the Centers for Disease Control and Prevention (CDC), more than 70% and 40% of American adults can be classified as overweight and obese, respectively. However, treatment strategies for dealing with cancer in obese patients have not advanced apart from lifestyle interventions. However, several signaling mechanisms have been proposed related to the inflammatory and metabolic impact of obese adipocytes in promoting cancer progression. However, a lack of mechanistic understanding led to no specific treatment strategies have been proposed which can specifically downregulate the key regulator by which obese adipocytes promote cancer progression. Thus, translational research initiatives are immediately needed to provide mechanistic justifications for these striking statistics in order to better serve this growing segment of the population. Here, we have shown the role of nanoscale physical communication between breast cancer cells and adipose tissue in promoting cancer progression. Using high-resolution optical and electron microscopy, we have found that breast cancer cells form nanoscale tubular communications with obese adipocytes. Moreover, the cancer cells hijack mitochondria from obese adipocytes. However, coculturing cancer cells and obese adipocytes in the Boyden chamber, allowing exosomal and paracrine communication but blocking direct physical communication, revealed negligible mitochondria transfer. This observation indicates an active transfer of mitochondria via nanotube. A significantly higher rate of mitochondria transfer from obese adipocytes to cancer cells has been observed than non-differentiated pre-adipocytes. The transfer of mitochondria results in an increase in the metabolism of the cancer cells. The mechanistic investigation of the nanotube formation revealed actin's presence as the major component in the nanotube. The exocyst-GTPase protein complex has a prime contribution in actin remodeling and nanotube formation. Inhibition of exocyst-GTPase using a pharmacological inhibitor results in a significant reduction in nanotube-mediated mitochondria transfer. Conclusion: Our goal is to (i) investigate the direct physical connection between adipocytes and cancer cells and the effect on cancer progression and (ii) eliminate mortality related to obesity-associated breast cancer severity by introducing a new therapeutic strategy. Here, we have shown how cancer cells communicate with obese adipocytes and receive additional metabolic power for proliferation. The in vivo investigation of the same is under progress. Citation Format: Tanmoy Saha, Balaaji Baanupriya Srinivasan, Shiladitya Sengupta. Involvement of nanoscale physical communication in obesity-associated breast cancer severity [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 3935.

Abstract 6788: Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models

Abstract Robust techniques are needed to screen the anti-tumor activity of antibody-drug conjugates (ADCs) in vitro. Traditional monolayer models provide a cost-effective and scalable option but lack many features of the tumor microenvironment, such as complex cell-cell and cell-extracellular matrix interactions. Tumor spheroids afford a more translational model for quantification of ADCs. Here, spheroids are used to compare the mechanism of action (MoA) of anti-HER2 ADCs. Single spheroids were formed from mono- or co-cultures of HER2 over-expressing cancer cells and HER2 negative control cells. Incucyte® Nuclight Green Lentivirus was used to label HER2 positive BT474 cells, whilst HER2 negative MDA-MB-231 cells were left unlabeled. Antibodies were added after 72 hours of spheroid formation in 96-well ultra-low attachment plates. Brightfield and fluorescence images were captured every 3 hours using the Incucyte® Live-Cell Analysis System and quantified, for metrics such as spheroid size and fluorescence intensity, to give an indication of ADC cytotoxicity. Co-culture spheroids were dissociated at assay endpoint and the remaining proportion of green and unlabeled cells was measured using the iQue® Advanced Flow Cytometry Platform. The area of the BT474 mono-culture spheroids decreased in a concentration-dependent manner in the presence of both ADCs tested (trastuzumab emtansine and trastuzumab deruxtecan), indicating increased cytotoxicity. In contrast, only the highest concentration of trastuzumab, which is the monoclonal antibody backbone on which they are based, resulted in a reduction in spheroid size. This displays the increased anti-tumor activity due to the addition of the ADC payloads. In the co-culture model, only the trastuzumab deruxtecan induced a reduction in spheroid area, with the size of the spheroids in the presence of trastuzumab emtansine and trastuzumab remaining comparable to control levels. Analysis of the spheroid composition using flow cytometry showed that for the BT474 cells, again, percentage cell death compared to the IgG control was greater for the two ADCs, at 96.4% and 97.2%, respectively. This death was greater than with trastuzumab, which induced 46.4% cell death. Unlike in the mono-culture assays, a high level of death of the HER2 low expressing cells was also seen in the co-culture in the presence of trastuzumab deruxtecan (94.1% death compared to control). This displays the unique bystander activity of trastuzumab deruxtecan, which has led to its approval as a treatment for both HER2 high and HER2 low breast cancers. These data display how the Incucyte® Live-Cell Analysis System combined with iQue® Advanced Flow Cytometry can be leveraged to provide comprehensive assessment of the in vitro function of ADCs and how it can enable differences in their MoAs to be distinguished in 3D spheroid models. Citation Format: Kirsty McBain, Kalpana Barnes, Nicola Bevan. Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models [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 6788.

Abstract 2893: Inhibition of ADAMTS4 and 5 to regulate the tumor microenvironment

Abstract Background: Triple-negative breast cancer accounts for approximately 10~15% of all breast cancers and is characterized by the absence of ER, PR, and HER2 receptors, making targeted therapies challenging due to the lack of biomarkers. ADAMTSs (ADAM metallopeptidase with thrombospondin) are complex extracellular proteases known for their roles in facilitating cancer cell proliferation and safeguarding tumor functions. This enzyme is secreted by cancer cells and various immune cells, influencing the tumor microenvironment. ADAMTSs modulate cellular adhesion, migration, proliferation, and vascular development by cleaving components of the extracellular matrix and regulatory factors such as chemokines and cytokines. Our aim is to positively manipulate the tumor microenvironment of triple-negative breast cancer by inhibiting ADAMTSs, presenting a new strategy for treating this challenging type of breast cancer that lacks targeted therapeutic options. Results: To confirm the anticancer effects of a protease inhibitor concurrently targeting ADAMTS4 and 5, a co-culture of triple-negative breast cancer cells (MDA-MB-157) and THP-1 was established to artificially create a tumor microenvironment. Subsequently, a wound healing assay was conducted, revealing that treatment with the ADAMTS4 and 5 dual inhibitor effectively inhibited cancer cell migration. This observed anticancer effect is attributed to the inhibition of CXCL16 secretion by the ADAMTS4 and 5 dual inhibitor. To verify the anti-tumor mechanisms of ADAMTS4 and 5, MDA-MB-157 cells, a triple-negative breast cancer cell line, were treated with IFNγ and TNFα to promote CXCL16 secretion artificially. Upon treatment with varying concentrations of the ADAMTS4,5 dual inhibitor, it was confirmed that the secretion of CXCL16 was inhibited in a dose-dependent manner. In a mouse model with transplanted 4T1 breast cancer cells, treatment with the ADAMTS4,5 dual inhibitor resulted in the suppression of CXCL16 secretion, leading to an increase in M1 macrophages. Additionally, it was observed that the inhibitor induced CD8+ T cell activation. These regulation and changes of immune cells through inhibition of ADAMTS4 and 5 control the TME to resist tumor cells. Conclusion: ADAMTSs are known as protein-degrading enzymes responsible for the degradation, modification, and regulation of proteins. Recent research has revealed that ADAMTSs play a crucial role in the proliferation and survival of cancer. Activation of ADAMTS4 and 5 has been shown to promote the secretion of CXCL16, leading to the differentiation of macrophages into tumor-associated macrophages. Specifically, ADAMTS4 and 5 convert membrane-bound CXCL16 into a soluble form, exacerbating the tumor microenvironment of triple-negative breast cancer. Therefore, inhibiting ADAM4,5 is intended to change or maintain TME in a suitable state for anti-cancer therapy, which could lead to a new strategy for anti-cancer therapy. Citation Format: Cham han Lee, Seong Keun Kim, Sung Moo Kim, Young Sang Kim, Sang Hyeob Lee, Seong Yun Ha, Yun Sun Lee, Jin Yi Yang. Inhibition of ADAMTS4 and 5 to regulate 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 2893.

Abstract 2895: ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts

Abstract Introduction: Pancreatic cancer (PDAC) patients have poor prognosis partly due to excessive activity of cancer-associated fibroblasts (CAFs). CAFs drive the fibrosis that causes excessive type III collagen and extracellular matrix deposition that in turn reduces drug response resulting in poor survival. In support, high levels of the type III collagen serum biomarker PRO-C3 correlates with poor survival in PDAC. TGF-β is thought to be the main driver of PRO-C3 and tumor fibrosis. Cytokines such as Interleukin 1 (IL-1) play a key role in the pancreatic tumor microenvironment and may play a role in tumor fibrosis as well. In this study, we first investigated the potential of IL-1 in activating fibroblasts to drive fibrosis and produce PRO-C3. Subsequently, we established a co-culture of pancreatic cancer cells and pancreatic CAFs to investigate the anti-fibrotic properties of nadunolimab, an antibody that blocks IL-1 signaling by targeting IL-1 Receptor Accessory Protein (IL1RAP). Nadunolimab is currently in phase I/IIa clinical development for treatment of pancreatic cancer (CANFOUR, NCT03267316). Methods: Human primary pancreatic cancer-associated fibroblasts (CAFs) were cultured in Ficoll-media (Scar-in-a-jar, SiaJ) supplemented with TGF-β, IL-1α or IL-1β. The fibrotic activity of the fibroblasts was investigated by measuring the formation of type III collagen (PRO-C3) at days 3, 6, 9 and 12. Then, pancreatic cancer cells (BxPc3) and pancreatic CAFs were cultured either alone or in a co-culture. Nadunolimab or isotype control were added at the start of these cultures and supernatants were collected after three days. The level of PRO-C3 was measured by ELISA. Results: Both IL-1α or IL-1β were equipotent to TGF-β in inducing PRO-C3 in the SiaJ monoculture, indicating that IL-1 is pro-fibrotic. In addition, when cancer cells and CAFs were co-cultured, PRO-C3 levels increased compared to single-cell cultures (1.5-6-fold). When co-cultures were treated with nadunolimab, the induction of PRO-C3 was blocked to levels similar to monocultures, whereas the isotype control had no effect on PRO-C3 levels. Conclusion: IL-1 activated fibroblasts and induced type III collagen formation (PRO-C3), suggesting that IL-1 is a driver of tumor fibrosis. In support, pancreatic tumor cells induced collagen type III formation (PRO-C3) in pancreatic CAFs and blockade of IL1RAP with nadunolimab inhibited this collagen formation. Thus, nadunolimab may have anti-fibrotic properties and PRO-C3 could potentially be used for prognostic/predictive enrichment and as a pharmacodynamic marker in future studies evaluating anti-IL-1 modalities in PDAC. Citation Format: Neel I. Nissen, Nils Hansen, Elin Jaensson Gyllenbäck, Camilla R. Millrud, Marcus Järås, David Liberg, Morten A. Karsdal, Nicholas Willumsen. ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts [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 2895.