Patient-derived Cancer-associated Fibroblasts Research Articles

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [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 PO2-24-06.

NNT-AS1 in CAFs-derived exosomes promotes progression and glucose metabolism through miR-889-3p/HIF-1α in pancreatic adenocarcinoma

It is metabolic and signaling crosstalk between stromal cells and tumors in the tumor microenvironment, which influences several aspects of tumor formation and drug resistance, including metabolic reprogramming. Despite considerable findings linking lncRNAs in HIF-1-related regulatory networks to cancer cell, little emphasis has been given to the role in communication between cancer-associated fibroblasts (CAFs) and tumor cells. Previously, we observed that NNT-AS1 was substantially expressed in CAFs cells and CAFs exosomes, and subsequently investigated the influence of CAFs exosomal NNT-AS1 on glucose metabolism, proliferation, and metastasis of pancreatic ductal adenocarcinoma (PDAC) cells. Transmission electron microscopy was used to examine exosomes secreted by PDAC patient-derived CAFs. qRT-PCR was used to evaluate the expression of NNT-AS1, miR-889-3p, and HIF-1. The role of CAFs-derived exosomal NNT-AS1 in PDAC cell progression and metabolism have been identified. Dual luciferase reporter assays examined the binding between NNT-AS1, miR-889-3p, and HIF-1. After PDAC cells co-culture exosomes secreted by CAFs, we found that they alter glucose metabolism, proliferation, and metastasis. In PDAC cells, CAF-derived exosomal lncRNA NNT-AS1 acted as a molecular sponge for miR-889-3p. Furthermore, HIF-1 could be targeted by miR-889-3p and was controlled by NNT-AS1. This study explores the mechanism by which NNT-AS1 influences the interaction of CAFs on glycolytic remodeling, proliferation, and metastasis of tumor cells through regulating miR-889-3p/HIF-1α, which also helps discover new clinical treatment targets for PDAC.

Abstract 5500: Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain metastasis

Abstract Introduction: Brain metastasis (BM) remains incurable with a dismal 2-year survival of less than 2%. BM are surrounded by a complex tumor microenvironment (TME) comprised of a meshwork of extracellular matrix (ECM) proteins and an assemblage of cell types including cancer-associated fibroblasts (CAFs), the most prominent cell type within the TME. We have previously shown that patient-derived BM CAFs can either support or inhibit tumor growth in vivo, suggesting that BM CAFs are heterogenous. Yet, there remains a dearth of data on the functional and mechanistic underpinnings of CAFs in the BM TME. Methods: To explore both the molecular profiles of CAFs and their heterogeneity in the BM TME, we performed a single cell Multiome (RNA and Assay for Transposase-Accessible Chromatin (ATAC)) sequencing (scMultiome-seq) analysis of 9 human BM tissue samples and 4 BM patient-derived CAF cell lines, from both lung and breast primary cancers. Next, we investigated the tumor supportive or inhibitory capabilities of BM CAF cell lines on the growth of BM tumor cells in vitro. Results: ScMultiome-seq analysis of BM tissue samples identified 4 distinct CAF subpopulations, that we termed desmoplastic, immune, contractile, and neural CAFs, in the BM TME. These CAF subpopulations are respectively involved in either regulating the ECM organization, orchestrating the neuroimmune response, triggering tissue contraction or regulating neural components upon BM growth. CellPhoneDB analysis revealed that CAFs had more cell-cell interactions with other cells than any other cell types in the BM TME. Pseudotime analysis demonstrated the potential cell of origin of each BM CAF subpopulation and the cell state transition they undergo. We observed that the end cell state of the desmoplastic CAFs expressed high levels of immunoglobulin superfamily containing leucine rich repeat (ISLR). ISLR was also observed to be highly expressed in a BM patient-derived CAF cell line which was previously reported by our lab to restrain tumor growth in vivo, suggesting that desmoplastic CAFs with high ISLR expression (ISLR-CAFs) might be tumor inhibitory. ScMultiome-seq analysis of BM CAF cell lines validated that tumor inhibitory CAF cell lines were primarily of the desmoplastic ISLR-CAF subpopulation. Conditioned media obtained from ISLR-CAFs containing high levels of ISLR inhibited tumor cell viability in vitro compared to controls. Conclusion: Our study is the first to describe the heterogeneity of CAFs and their potential central roles in the BM TME. We also identify that CAF subpopulations are dynamic and can transition to tumor inhibitory cell states, such as ISLR-CAFs. These data have potential to help design therapeutic strategies that selectively target tumor supportive CAFs whilst protecting tumor inhibitory ones. Citation Format: Thomas Simon, David N. Buckley, Ben Y. Tew, Matthew Salomon, Zeyi Yang, Gerald C. Gooden, Steven A. Toms, Gabriel Zada, Bodour Salhia. Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain 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 5500.

Abstract 217: Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity

Abstract Background: Cancer-associated fibroblasts (CAFs) are heterogeneous, with CAF subtypes being localized in spatially defined areas of tumors. However, it remains unclear which CAF subsets specifically play a role in fundamental processes such as tumor growth, invasion, and response to therapy. This study aims to establish a novel in vitro model that recapitulates the in vivo tumor-stroma spatial relationship to investigate CAF heterogeneity and its impact on tumor cell growth in non-small cell lung cancer (NSCLC). Methods: The in vitro model was established using NSCLC patient-derived CAFs and tumor organoids as a 6:1 ratio. Tumor cells originating from xenograft-derived organoids (XDOs) were embedded in a hydrogel containing CAFs (Day 0) and co-cultured for 5 days. Confocal microscopy was utilized to characterize and measure the proliferation of the XDO tumor cells in the co-culture setup, and the proliferation rate was defined as differences in tumor spheroid area between Day 1 and 5. Single-cell RNA-sequencing (scRNAseq) and gene set enrichment analysis (GSEA) were then used to assess the gene expression and signaling pathways dominant in mono- and co-culture to characterize CAF heterogeneity in this model. Results: Initially, the culture media formulations and the matrix for the growth of both CAFs and tumor cells were screened to assess the compatibility of different culture elements. Both CAFs and XDO tumor cells proliferated well in reduced organoid media (M27RD). This reduced medium and the novel matrix composition (50% Matrigel/50% Rat Tail type I collagen) also allowed for maintaining CAF contractility and XDO proliferation. Utilizing these harmonized media and matrix conditions, a spatially defined co-culture model, which incorporated XDOs as a central cluster surrounded by CAFs, was generated. In confocal microscopy, the proliferation rate of tumor cells was significantly higher in co-cultures with CAFs (adjusted p-value < 0.01), while not significant in mono-culture of tumor cells. The GSEA using the Hallmark gene sets on scRNAseq data generated in three co-cultures identified the heterogeneity among CAFs: two CAFs showed significantly higher effect on tumor cell proliferation with the most highly differential expressed genes (DEGs) enriched with inflammation and mitosis and the top high DEGs associated with epithelial-mesenchymal transition, while the other CAF has no effect. Conclusion: The harmonized co-culture conditions for NSCLC organoids and CAFs enabled the characterization of both cell phenotypes and the building of an informed model architecture to study tumor-stroma communication. The presence of CAFs increased tumor cell growth with enrichment in secreted proteins involved in epithelial-mesenchymal transition. Citation Format: Cheol-Kyu Park, Roya Navab, Elisa D'Arcangelo, Quan Li, Hiroyuki Ogawa, Nikolina Radulovich, Nhu-An Pham, Ming-Sound Tsao. Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity [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 217.

Abstract 636: Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts

Abstract The tumor microenvironment (TME) is well recognized for its role in tumor progression, chemoresistance, and recurrence. The TME consists of various components, such as extracellular matrix (ECM), chemical and mechanical cues, as well as stromal cells and their interactions. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have drawn a lot of attention. It has been reported that CAFs not only directly interact with cancer cells via surface proteins and cytokines, but also influence their behaviors by remodeling the ECM. However, due to their heterogenous characteristics and diverse functions, the influence of tumor-CAF interactions on drug response is not fully understood. Here, we present a coculture model for investigating CAF effects on patient-derived colorectal tumor organoids (CTOs) growth, differentiation, and drug response. It consists of normal colon fibroblasts, CCD18-Co, or patient-derived CAFs seeded with CTOs in a mixed hydrogel containing 90% basement membrane extract (BME) and 10% collagen type I. Media conditions were optimized (1:1 mix of CTO culture media plus reduced media excluding growth factors and inhibitors) to achieve similar organoid sizes in the monoculture and coculture conditions at the onset of drug testing. To support higher throughput investigations, our workflow incorporates a liquid handler for automated CTO seeding, media and drug exchange, and an automated microscope with a robotic arm for multi-plate imaging. Briefly, single cell CTOs and fibroblasts were seeded at a 1:1 ratio (3000 total cells/well) in 96-well plates. After 4 days in culture, anti-cancer drugs were administrated in reduced media to the samples. Brightfield images were acquired on days 0, 3, and 5 after drug administration. The collected images were analyzed with a neural network (NN) to dynamically monitor the growth and viability of individual organoids. The CellTiter-Glo assay was performed on day 5 to quantify the total CTO cell viability. The normalized drug response (NDR) was calculated from the NN processed images to determine the drug response. An immunostaining panel, which includes protein markers such as CK20, Ki67, α-SMA, Vimentin, and FAP, was used to evaluate the fibroblast phenotypes and CTO differentiation. Additionally, the cultures were subjected to fluorescent lifetime imaging microscopy (FLIM), a label-free imaging technique, to measure the metabolic adaptations of each cell population. Preliminary studies revealed a CAF-dependent increase in CTO growth and glycolytic signatures in the co-culture setting; however, minimal effects on drug response to common chemotherapy agents such as 5-fluorouracil and irinotecan were observed. In the future, we will use this integrated pipeline as a screening method to identify new treatment strategies aimed at targeting the stromal microenvironment in colorectal cancer. Citation Format: Yuyuan Zhou, Yu-Kai Huang, Emma J. Fong, Elizabeth Elton, Erin McManus, Brandon Choi, Seungil Kim, Shannon M. Mumenthaler. Coculture model for investigating cellular interactions between colorectal tumor organoids and 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 636.

Bioengineered Hydrogels Recapitulate Fibroblast Heterogeneity in Cancer.

Recently mapped transcriptomic landscapes reveal the extent of heterogeneity in cancer-associated fibroblasts (CAFs) beyond previously established single-gene markers. Functional analyses of individual CAF subsets within the tumor microenvironment are critical to develop more accurate CAF-targeting therapeutic strategies. However, there is a lack of robust preclinical models that reflect this heterogeneity in vitro. In this study, single-cell RNA sequencing datasets acquired from head and neck squamous cell carcinoma tissues to predict microenvironmental and cellular features governing individual CAF subsets are leveraged. Some of these features are then incorporated into a tunable hyaluronan-based hydrogel system to culture patient-derived CAFs. Control over hydrogel degradability and integrin adhesiveness enabled derivation of the predominant myofibroblastic and inflammatory CAF subsets, as shown through changes in cell morphology and transcriptomic profiles. Last, using these hydrogel-cultured CAFs, microtubule dynamics are identified, but not actomyosin contractility, as a key mediator of CAF plasticity. The recapitulation of CAF heterogeneity in vitro using defined hydrogels presents unique opportunities for advancing the understanding of CAF biology and evaluation of CAF-targeting therapeutics.

Viscoelastic stiffening of gelatin hydrogels for dynamic culture of pancreatic cancer spheroids

The tumor microenvironment (TME) in pancreatic adenocarcinoma (PDAC) is a complex milieu of cellular and non-cellular components. Pancreatic cancer cells (PCC) and cancer-associated fibroblasts (CAF) are two major cell types in PDAC TME, whereas the non-cellular components are enriched with extracellular matrices (ECM) that contribute to high stiffness and fast stress-relaxation. Previous studies have suggested that higher matrix rigidity promoted aggressive phenotypes of tumors, including PDAC. However, the effects of dynamic viscoelastic matrix properties on cancer cell fate remain largely unexplored. The focus of this work was to understand the effects of such dynamic matrix properties on PDAC cell behaviors, particularly in the context of PCC/CAF co-culture. To this end, we engineered gelatin-norbornene (GelNB) based hydrogels with a built-in mechanism for simultaneously increasing matrix elastic modulus and viscoelasticity. Two GelNB-based macromers, namely GelNB-hydroxyphenylacetic acid (GelNB-HPA) and GelNB-boronic acid (GelNB-BA), were modularly mixed and crosslinked with 4-arm poly(ethylene glycol)-thiol (PEG4SH) to form elastic hydrogels. Treating the hybrid hydrogels with tyrosinase not only increased the elastic moduli of the gels (due to HPA dimerization) but also concurrently produced 1,2-diols that formed reversible boronic acid-diol bonding with the BA groups on GelNB-BA. We employed patient-derived CAF and a PCC cell line COLO-357 to demonstrate the effect of increasing matrix stiffness and viscoelasticity on CAF and PCC cell fate. Our results indicated that in the stiffened environment, PCC underwent epithelial-mesenchymal transition. In the co-culture PCC and CAF spheroid, CAF enhanced PCC spreading and stimulated collagen 1 production. Through mRNA-sequencing, we further showed that stiffened matrices, regardless of the degree of stress-relaxation, heightened the malignant phenotype of PDAC cells. Statement of significanceThe pancreatic cancer microenvironment is a complex milieu composed of various cell types and extracellular matrices. It has been suggested that stiffer matrices could promote aggressive behavior in pancreatic cancer, but the effect of dynamic stiffening and matrix stress-relaxation on cancer cell fate remains largely undefined. This study aimed to explore the impact of dynamic changes in matrix viscoelasticity on pancreatic ductal adenocarcinoma (PDAC) cell behavior by developing a hydrogel system capable of simultaneously increasing stiffness and stress-relaxation on demand. This is achieved by crosslinking two gelatin-based macromers through orthogonal thiol-norbornene photochemistry and post-gelation stiffening with mushroom tyrosinase. The results revealed that higher matrix stiffness, regardless of the degree of stress relaxation, exacerbated the malignant characteristics of PDAC cells.

Abstract B062: CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to therapy. Stromal cancer-associated fibroblasts (CAFs) play a key role in promoting tumor progression and chemoresistance, by creating a fibrotic microenvironment that impedes drug access and feeds PDAC cells nutrients and pro-tumor signals. CRO-67 (Patent PCT/AU2023/050505) is a novel drug developed with Noxopharm Ltd using a rational medicinal chemistry design to improve bioavailability of chromans which have potent anti-cancer activity. Using our patient-derived PDAC tumor in a dish model (explants; maintain multicellular architecture and fibrosis) we previously showed that CRO-67 has both anti-tumor and CAF reprogramming capacity in 4 patient explants [Cancer Res (2022) 82 (22_Supplement):C073]. Aims: 1) To expand evaluation of CRO-67 in additional patient-derived PDAC tumor explants given PDAC heterogeneity. 2) Assess the effect of CRO-67 on PDAC cell and CAF function. 3) Validate the therapeutic potential of CRO-67 on PDAC growth in vivo. Methods: 1) PDAC tumor samples collected from 7 patients undergoing pancreatic resection. Tumor explants (1–2mm diameter) were cultured on gelatin sponges, treated with CRO-67 (0–50μg/mL) every 3 days and fixed on day 12. Therapeutic response assessed by immunohistochemistry for cytokeratin (PDAC cells), α-smooth muscle actin (CAFs), bromodeoxyuridine (proliferation) and TUNEL (cell death). 2) PDAC cells (MiaPaCa-2) and patient-derived CAFs were treated with CRO-67 (1.5μM) for 48h before proliferation analysis (IncuCYTE S3) and for 24h before apoptosis (Annexin V/DAPI staining; flow cytometry) and cell cycle (DAPI staining; flow cytometry) analysis. 3) Subcutaneous PDAC (BxPC3 human cells) tumors in mice were treated with CRO-67 (2.5mg/kg Intraperitoneally, twice a day) for 21 days and tumor volume measured (calipers). Results: 1) CRO-67 treatment decreased tumor and CAF cell frequency in 6/6 (no quantifiable tumor in patient 7 explants) and 7/7 patient PDAC explants, respectively. It also decreased explant cell proliferation and increased cell death versus controls. 2) CRO-67 completely abolished proliferation of MiaPaCa-2 and reduced the average growth rate of CAFs by 81.3±8.8% (p=0.0025; n=5), increased apoptosis in MiaPaCa-2 by 455.7±21.4% (p=0.003; n=3) and in CAFs by 172.6±13.1% (p=0.0067; n=5) and increased the fraction of cells in G2/M cell cycle phase by 163.8±12.8% (p<0.0001; n=3) in MiaPaCa-2 and by 74.6±8.6% (p=0.0015; n=5) in CAFs versus controls. 3) CRO-67 reduced PDAC tumors in vivo by 56.7±6.6% (p=0.0013; n=9 mice/group) versus controls. Conclusions: CRO-67: 1) reduced PDAC cells and CAFs in human PDAC explants via reduced proliferation and increased death; 2) reduced proliferation of PDAC cells and CAFs in vitro, increased apoptosis and disrupted cell cycle progression through G2/M phase; 3) reduced PDAC tumor growth in vivo. Implication: We predict CRO-67 is a potential ‘dual cell’ therapy with direct anti-tumor effects and CAF reprogramming capacity, warranting further investigation in vivo. Citation Format: Keilah Garcia Netto, Shannon Chiang, John Kokkinos, Koroush S. Haghighi, Aparna Raina, Omali Pitiyarachchi, Janet Youkhana, Quach Truong, Daniel Wenholz, Xiang Li, Olivier Laczka, Naresh Kumar, John Wilkinson, David Goldstein, George Sharbeen, Phoebe A. Phillips. CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B062.

Protocol for high throughput 3D drug screening of patient derived melanoma and renal cell carcinoma

High Throughput Screening (HTS) with 3D cell models is possible thanks to the recent progress and development in 3D cell culture technologies. Results from multiple studies have demonstrated different drug responses between 2D and 3D cell culture. It is now widely accepted that 3D cell models more accurately represent the physiologic conditions of tumors over 2D cell models. However, there is still a need for more accurate tests that are scalable and better imitate the complex conditions in living tissues. Here, we describe ultrahigh throughput 3D methods of drug response profiling in patient derived primary tumors including melanoma as well as renal cell carcinoma that were tested against the NCI oncologic set of FDA approved drugs. We also tested their autologous patient derived cancer associated fibroblasts, varied the in-vitro conditions using matrix vs matrix free methods and completed this in both 3D vs 2D rendered cancer cells. The result indicates a heterologous response to the drugs based on their genetic background, but not on their maintenance condition. Here, we present the methods and supporting results of the HTS efforts using these 3D of organoids derived from patients. This demonstrated the possibility of using patient derived 3D cells for HTS and expands on our screening capabilities for testing other types of cancer using clinically approved anti-cancer agents to find drugs for potential off label use.

A Synergetic Approach Utilizing Nanotechnology, Chemotherapy, and Radiotherapy for Pancreatic Cancer Treatment

<img src=” https://s3.amazonaws.com/production.scholastica/article/90447/large/prnano_1112023ga.jpg?1700670543”> The objective of this study was to assess the anticancer effectiveness of gold nanoparticles (GNPs) and lipid-encapsulated docetaxel prodrug (LNPDTX-P) with radiotherapy (RT). The study utilized a co-culture spheroid model comprising MIA PaCa-2 cancer cells and patient-derived cancer-associated fibroblasts (CAF-98) to mimic pancreatic cancer conditions. The spheroids underwent treatment with GNPs (7.5 μg/mL), LNPDTX-P (99 nM of DTX pro-drug), and 2 Gy of RT. Cell viability of the spheroids was evaluated using the CellTiter-Glo 3D assay. At the same time, DNA double-strand breaks (DSBs) were assessed by examining the expression of the DNA damage marker 53BP1 through an immunofluorescence assay. Alt-hough GNPs/RT and RT/LNPDTX-P showed a reduction in spheroid size and an apparent in-crease in DNA DSB damage, the combination of the two nanoparticles, GNPs, and LNPDTX-P, with RT, significantly enhanced the anticancer efficacy, resulting in a 28% decrease in spheroid size and an estimated 39% increase in DNA DSB. The combination of GNPs and LNPDTX-P with RT showed a synergetic effect due to their radiosensitizing properties, improving the ther-apeutic efficacy of each treatment modality alone. This triple modality offers a hopeful strategy to enhance cancer treatment efficacy while reducing adverse effects.

EDIL3 as an Angiogenic Target of Immune Exclusion Following Checkpoint Blockade.

Immune checkpoint blockade (ICB) has become the standard of care for several solid tumors. Multiple combinatorial approaches have been studied to improve therapeutic efficacy. The combination of antiangiogenic agents and ICB has demonstrated efficacy in several cancers. To improve the mechanistic understanding of synergies with these treatment modalities, we performed screens of sera from long-term responding patients treated with ipilimumab and bevacizumab. We discovered a high-titer antibody response against EGF-like repeats and discoidin I-like domains protein 3 (EDIL3) that correlated with favorable clinical outcomes. EDIL3 is an extracellular protein, previously identified as a marker of poor prognosis in various malignancies. Our Tumor Immune Dysfunction and Exclusion analysis predicted that EDIL3 was associated with immune exclusion signatures for cytotoxic immune cell infiltration and nonresponse to ICB. Cancer-associated fibroblasts (CAF) were predicted as the source of EDIL3 in immune exclusion-related cells. Furthermore, The Cancer Genome Atlas Skin Cutaneous Melanoma (TCGA-SKCM) and CheckMate 064 data analyses correlated high levels of EDIL3 with increased pan-fibroblast TGFβ response, enrichment of angiogenic signatures, and induction of epithelial-to-mesenchymal transition. Our in vitro studies validated EDIL3 overexpression and TGFβ regulation in patient-derived CAFs. In pretreatment serum samples from patients, circulating levels of EDIL3 were associated with circulating levels of VEGF, and like VEGF, EDIL3 increased the angiogenic abilities of patient-derived tumor endothelial cells (TEC). Mechanistically, three-dimensional microfluidic cultures and two-dimensional transmigration assays with TEC endorsed EDIL3-mediated disruption of the lymphocyte function-associated antigen-1 (LFA-1)-ICAM-1 interaction as a possible means of T-cell exclusion. We propose EDIL3 as a potential target for improving the transendothelial migration of immune cells and efficacy of ICB therapy.

Utilizing Gold Nanoparticles as Prospective Radiosensitizers in 3D Radioresistant Pancreatic Co-Culture Model.

Pancreatic cancer stands among the deadliest forms of cancer, and the existing treatments fall short of providing adequate efficacy. Novel and more effective treatment approaches are urgently required to address this critical medical challenge. In this study, we aimed to evaluate the anti-cancer efficacy of gold nanoparticles (GNPs) in combination with radiotherapy (RT). A 3D pancreatic cancer co-culture spheroid model of MIA PaCa-2 cancer cells and patient-derived cancer-associated fibroblasts (CAF-98) was used. The spheroids were treated with GNPs (7.5 μg/mL) and 2 Gy of RT. The spheroids' cell viability was assessed through the CellTiter-Glo 3D assay, and an immunofluorescence assay was used to assess the DNA DSBs via the expression of the DNA damage marker 53BP1. Co-culture samples showed a 10.8% (p < 0.05) increase in proliferation and a 13.0% (p < 0.05) decrease in DNA DSB when compared to monoculture samples, However, they displayed a 175% (p < 0.001) increase in GNPs uptake when compared to monoculture spheroids. Using GNPs/RT, we were able to show a significant reduction of 6.2% (p < 0.05) in spheroid size and an increase of 14.3% (p < 0.05) in DNA DSB damage in co-culture samples. The combination of GNPs with RT demonstrated remarkable radiosensitization effects, representing a promising approach to enhance cancer treatment efficacy. These effects were particularly noteworthy in the more treatment-resistant co-culture spheroid model.

The Impact of Cancer-Associated Fibroblasts on Drug Resistance, Stemness, and Epithelial-Mesenchymal Transition in Bladder Cancer: A Comparison between Recurrent and Non-Recurrent Patient-Derived CAFs

This study comparatively evaluated the possible effects of recurrent and non-recurrent patient-derived Cancer-Associated Fibroblasts (CAFs-R and -NR) on the bladder cancer cell line, EJ138. Both groups of CAFs increased cisplatin resistance and altered cell cycle distribution alongside induced resistance to apoptosis. Later, the scratch assay confirmed the cell migration-inducing effects of CAFs on cells. Nonetheless, only CAFs-R managed to increase sphere-formation and clonogenic levels in EJ138 cells, which were later validated by upregulating pluripotency transcription factors. Besides, CAFs-R also affected the expression levels of some of the EMT markers. Our study suggests that CAFs-R had stronger pro-tumorigenic effects on EJ138 cells.

RAGE inhibition blunts insulin-induced oncogenic signals in breast cancer

The receptor for advanced glycation end products (RAGE) is implicated in diabetes and obesity complications, as well as in breast cancer (BC). Herein, we evaluated whether RAGE contributes to the oncogenic actions of Insulin, which plays a key role in BC progression particularly in obese and diabetic patients. Analysis of the publicly available METABRIC study, which collects gene expression and clinical data from a large cohort (n = 1904) of BC patients, revealed that RAGE and the Insulin Receptor (IR) are co-expressed and associated with negative prognostic parameters. In MCF-7, ZR75 and 4T1 BC cells, as well as in patient-derived Cancer-Associated Fibroblasts, the pharmacological inhibition of RAGE as well as its genetic depletion interfered with Insulin-induced activation of the oncogenic pathway IR/IRS1/AKT/CD1. Mechanistically, IR and RAGE directly interacted upon Insulin stimulation, as shown by in situ proximity ligation assays and coimmunoprecipitation studies. Of note, RAGE inhibition halted the activation of both IR and insulin like growth factor 1 receptor (IGF-1R), as demonstrated in MCF-7 cells KO for the IR and the IGF-1R gene via CRISPR-cas9 technology. An unbiased label-free proteomic analysis uncovered proteins and predicted pathways affected by RAGE inhibition in Insulin-stimulated BC cells. Biologically, RAGE inhibition reduced cell proliferation, migration, and patient-derived mammosphere formation triggered by Insulin. In vivo, the pharmacological inhibition of RAGE halted Insulin-induced tumor growth, without affecting blood glucose homeostasis. Together, our findings suggest that targeting RAGE may represent an appealing opportunity to blunt Insulin-induced oncogenic signaling in BC.

Isolation and characterization of cancer-associated fibroblasts in the tumor microenvironment of hepatocellular carcinoma.

Cancer-associated fibroblasts (CAFs) play an immunosuppressive role in the tumor microenvironment (TME) of human cancers; however, their characteristics and role in hepatocellular carcinoma (HCC) remain to be elucidated. Nine tumor and surrounding liver tissue samples from patients with HCC who underwent surgery were used to isolate patient-derived CAFs. Cell morphology was observed using an optical microscope after culture, and cell phenotypes were evaluated using flow cytometry and immunoblotting. Cytokines secreted by CAFs into culture medium were quantified using a multiplex cytokine assay. CAFs were abundant in the TME of HCC and were adjacent to immune cells. After culture, the CAFs and non-tumor fibroblasts exhibited spindle shapes. We observed a robust expression of alpha-smooth muscle actin and fibroblast activation protein in CAFs, whereas alpha-fetoprotein, epithelial cell adhesion molecule, platelet/endothelial cell adhesion molecule-1, and E-cadherin were not expressed in CAFs. Furthermore, CAFs showed high secretion of various cytokines, namely C-X-C motif chemokine ligand 12, interleukin (IL)-6, IL-8, and C-C motif chemokine ligand 2. CAFs are abundant in the TME of HCC and play a crucial role in tumor progression. These fibroblasts secrete cytokines that promote tumor growth and metastasis.

Targeting TGF beta receptor 1 in head and neck squamous cell carcinoma.

The transforming growth factor-Beta (TGF-ß) pathway may be involved in the radioresistance of head and neck squamous cell carcinoma (HNSCC). This study analyzed TGF-ß receptor 1 (TGFBR1) expression in HNSCC patients and evaluated the antineoplastic and radiosensitizing effects of vactosertib, a novel TGFBR1 inhibitor, in vitro. TGFBR1 expression was examined in HNSCC patients at the mRNA level in silico and the protein level by immunohistochemistry, including surgical specimens of primary tumors, matched lymph node metastasis, and recurrent disease. Furthermore, a novel small molecule TGFBR1 inhibitor was evaluated in HNSCC cell lines. Finally, an indirect coculture model using patient-derived cancer-associated fibroblasts was applied to mimic the tumor microenvironment. Patients with high TGFBR1 mRNA levels showed significantly worse overall survival in silico (OS, p = 0.024). At the protein level, an association between TGFBR1+ tumor and OS was observed for the subgroup with TGFBR1-stroma (p = 0.001). Those results prevailed in multivariable analysis. Inhibition of TGFBR1 showed antineoplastic effects in vitro. In combination with radiation, vactosertib showed synergistic effects. Our results indicate a high risk of death in tumorTGFBR1+|stromaTGFBR1- expressing patients. In vitro data suggest a potential radiosensitizing effect of TGFBR1 inhibition by vactosertib.

Abstract 5851: Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a classically heterogeneous tumor for which tumor cells comprise less than 40% of the total tumor mass. To better understand mechanisms of intercellular interactions driving tumor response or resistance to chemotherapy, it is crucial to account for the complex tumor microenvironment (TME). Cancer associated fibroblasts (CAFs) have been implicated as key drivers supporting mechanisms that both promote and restrain tumor growth. Using our model system that investigates interactions between CAFs and cancer cells, we show a novel intercellular interaction through VEGF signaling that drives proliferation. Methods: We interrogate intracellular crosstalk in the PDAC TME using a novel 3-dimensional (3D), patient-matched coculture system of patient-derived organoids (PDO) and CAFs, obtained from patients undergoing surgical resection. Molecular characterization is performed by profiling with a 1200 analyte multiplex ELISA screen, multiparameter flow cytometry, bulk RNA sequencing of CAF and PDO cocultures, imaging mass cytometry (IMC) on patient samples, and qPCR. Results: Secretome profiling of 7 patient-derived CAF lines demonstrated patient-specific heterogeneity in proteins such as: FGF-12, FGF3, and CD79a. Common CAF-derived signaling proteins with the potential to regulate intercellular crosstalk that were seen across all samples include CRP, IL-6R, R-Spondin and others. To better understand how factors change with intercellular interactions, we set up direct CAF-PDO cocultures over 4 days to identify global transcriptional changes using bulk RNAseq. Phenotypically, coculture drives gene expression changes in both PDO subtype (basal vs classical) and CAF subtype (prevalence of iCAFs vs myCAFs). We used these transcriptome data to identify pathways modulated by CAF-PDO crosstalk. Profiling coculture supernatant, we identified upregulation in VEGF secretion. In tandem, when we investigate cellular surface marker changes, we see upregulation of VEGFR2 on the surface of CAFs by flow cytometry. This interaction is accompanied by an increase in PDO proliferation seen in the coculture conditions. Ongoing experiments aim to investigate the impact of this relationship spatially using matched patient tissue. Conclusions: Interactions between cancer cells and CAFs compound the complexity of the biology of the TME and contribute to poor patient outcomes; therefore, we have put forth a model that better represents these interactions through patient matched PDO - CAF cocultures. Using this model, we demonstrate a novel interaction through VEGF that enhances tumor cell proliferation. We introduce a targeted approach to investigating the complex biology of the TME to inform the mechanisms driving cancer biology in individual patients that can also be used to develop novel therapeutic targets. Citation Format: Samantha Guinn, Joseph Tandurella, Jignasha Patel, Richard Burkhart, Jacquelyn Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5851.

Abstract 5864: An angiogenic target of immune exclusion with checkpoint blockade in melanoma

Abstract With the development of immune checkpoint blockade to treat a variety of malignancies, better predictive biomarkers as well as new targets to further improve anti-tumor immunity remains a need. Our understanding of the role of angiogenesis in immune regulation continues to improve, and combinations of anti-angiogenesis and immune checkpoint blockade demonstrate improved clinical efficacy, as well as mechanisms for improved immune cell trafficking. Tumor biopsies from a phase I study with 46 advanced melanoma patients treated with Ipilimumab and Bevacizumab combination (Ipi-Bev) therapy, revealed activated endothelium accompanied by lymphocyte infiltration. To discover possible targets involved in the observed clinical benefit, a serological screen was performed using human protein arrays with post-treatment plasma samples of long-term patients with clinical benefit to Ipi-Bev therapy (Wu et. al., 2017; Hodi et. al., 2014). EDIL3 (EGF Like Repeats and Discoidin Domains 3), a pro-angiogenic extracellular matrix protein, was identified. Humoral responses against EDIL3 were associated with favorable clinical outcomes including improved response rates (p=0.003) and overall survival (p=0.03) to Ipi-Bev therapy. The anti-EDIL3 response was also observed to be significantly (p=0.03) related to Ipi-Bev treatment when compared with patients treated with Ipilimumab alone, anti-PD1 therapy alone, or the combination of Ipilimumab and Nivolumab in a combined analysis. Transcriptome analyses of TCGA SKCM (n=469) and BMS Checkmate 064 trial (n=90) related upregulated EDIL3 expression with enrichment of EMT, TGFβ signaling in fibroblasts, and angiogenesis signatures. Interestingly, TIDE analysis demonstrated a role for EDIL3 in T cell exclusion and immunosuppressive cancer-associated fibroblasts (CAFs) as its source in the tumor microenvironment. EDIL3 was also predicted as a biomarker of non-response to immune checkpoint blockade treatment. In vitro studies confirmed the upregulation of EDIL3 expression and secretion by patient-derived CAFs along with its role in modulating TGFβ1 mediated EMT. Mechanistically, EDIL3 was found to interfere with leukocyte-endothelial interactions by disturbing LFA-1/ICAM1 mediated adhesion and inhibited T cell transmigration in 2-D and microfluidic 3-D coculture studies with patient-derived tumor endothelial cells. Our findings suggest that the humoral response against EDIL3 may serve as one of the underlying mechanisms involved in effectuating an excluded to an inflamed phenotype observed in responders of checkpoint blockade treatment. This study develops EDIL3 as a potential target to enhance cytotoxic immune responses and improve the outcomes of combination immune therapy. Studies are ongoing for targeted inhibition of EDIL3 with implications for therapeutic application to promote anti-tumor immunity. Citation Format: Saba Tabasum, Dinesh Thapa, Anita Giobbie-Hurder, Jason L. Weirather, Xinqi Wu, Marco Campisi, Xiaoyun Li, Jingjing Li, Michael P. Manos, David A. Barbie, F. Stephen Hodi. An angiogenic target of immune exclusion with checkpoint blockade in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5864.

Abstract 1338: Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling

Abstract Ovarian cancer (OC) is the most lethal gynecologic cancer and the 5th leading cause of cancer-related deaths among women in the US. One of the main reasons for the poor outcome is a high recurrence rate and development of chemoresistance. The standard care involves debulking surgery and carbo-taxol chemotherapy. While most patients respond well initially, a small fraction of OC cells survive - these persister cells are enriched in cancer stem cells (CSCs), and are capable of tumor initiation and cause relapse. Most studies on OC stem cells (OCSCs) have focused on their cell-intrinsic properties and not much is known about the role of the tumor microenvironment (TME) in regulating their growth, fate, and sustenance. Cancer-associated fibroblasts (CAFs) are a key constituent of the OC TME and have been reported to play important roles in regulating tumorigenesis, metastasis, and immune evasion. Moreover, the residual tumors following neo-adjuvant chemotherapy are highly fibrotic. Our studies using matched pre- and post-chemotherapy OC patient tumors revealed an enrichment in OCSCs and CAFs following chemotherapy. Using heterotypic 3D cocultures of patient-derived CAFs and OC cells and in vivo limiting dilution assay, we found that CAFs could induce OCSCs. CAFs stimulated symmetric division of OCSCs as well as dedifferentiation of some non-OCSCs. Using a novel interface interaction assay that we developed, we found that CAFs could influence only those OC cells that are in their immediate neighborhood. We have identified the underlying mechanism involving a non-canonical Wnt signaling pathway in the OCSCs induced by Wnt5a secreted by CAFs. Using functional rescue experiments, we determined that Wnt5a acted via its coreceptor ROR2 on the OCSCs that activated PKC and CREB1 to induce stemness. Inhibiting the CAF-OCSC crosstalk using a Wnt5a-specific inhibitor, Box5 abrogated the effects of CAFs on OCSCs both in vitro and in vivo. While Box5 treatment alone was sufficient to inhibit tumor growth in vivo, its combination with carboplatin sensitized tumors to carboplatin by eradicating residual OCSCs. CAFs are heterogenous and not all CAFs are capable of inducing OCSCs, similarly, only a subpopulation of OC cells can respond to CAFs and dedifferentiate into OCSCs. We performed single-cell RNA-seq using heterotypic 3D cocultures of patient-derived CAFs and OC cells, and characterized the pathways activated in Wnt5ahigh CAFs. Using trajectory analysis, we identified OC subpopulations that respond to CAF signals and become OCSCs. In summary, we have identified a novel mechanism by which a subpopulation of CAFs in the OC TME act as OCSC niche. Our research provides compelling evidence in support of targeting Wnt5a, in combination with the standard of care carbo-taxol chemotherapy, to prevent OC relapse and chemoresistance. Citation Format: Xue Xiao, Yiming Fang, David Pepin, Dmitriy Zamarin, Anirban K. Mitra. Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1338.

Patient-Derived Primary Cancer-Associated Fibroblasts Mediate Resistance to Anti-Angiogenic Drug in Ovarian Cancers.

Ovarian cancers rank first in both aggressiveness and dismal prognosis among gynecological neoplasms. The poor outcome is explained by the fact that most patients present with late-stage disease and progress through the first line of treatment. Ovarian neoplasms, especially epithelial ovarian cancers, are diagnosed at advanced/metastatic stages, often with a high angiogenesis index, one of the hallmarks of ovarian cancers with rapid progression and poor outcome as resistance to anti-angiogenic therapy develops. Despite therapy, the metastatic progression of aggressive ovarian cancer is a spectacularly selective function of tumor cells aided and abetted by the immune, mesenchymal and angiogenic components of the tumor microenvironment (TME) that enforces several pro-metastatic event(s) via direct and indirect interactions with stromal immune cells, cancer-associated fibroblasts (CAFs), and vascular endothelial cells. Since transdifferentiation of tumor endothelium is one of the major sources of CAFs, we hypothesized that ovarian CAF plays a critical role in resisting anti-angiogenic effects via direct crosstalk with endothelium and hence plays a direct role in the development of resistance to anti-angiogenic drugs. To test the hypothesis, we set up a hybrid ex vivo model for co-culture comprising Patient-Derived ex vivo primary CAFs from ovarian tumor samples and human umbilical vein endothelial cells (HUVEC). Patient-Derived CAFs were characterized by the mRNA and protein expression of positive (SMA, S100A4, TE-7, FAP-A, CD90/THY1), negative (EpCAM, CK 8,18, CD31, CD44, CD45), functional (PDGFRA, TGFB1, TGFB2, TGFRA) and immunological markers (PD-L1, PD-L2, PD-1) associated with CAFs by qRT-PCR, flow cytometry, Western blot, and ICC. Data from our HUVEC-on-CAF ex vivo Hybrid Co-Culture (HyCC) study demonstrate the pro-angiogenic effect of Patient-Derived ovarian CAFs by virtue of their ability to resist the effect of anti-angiogenic drugs, thereby aiding the development of resistance to anti-angiogenic drugs. Ascertaining direct experimental proof of the role of CAFs in developing resistance to specific anti-angiogenic drugs will provide an opportunity to investigate new drugs for counteracting CAF resistance and "normalizing/re-educating" TME in aggressive ovarian cancers. Our data provide a unique experimental tool for the personalized testing of anti-angiogenic drugs, positively predicting the development of future resistance to anti-angiogenic drugs well before it is clinically encountered in patients.