Rab31-dependent regulation of transforming growth factor ß expression in breast cancer cells.

BACKGROUND: The small GTP-binding protein Rab31 plays an important role in the modulation of tumor biological-relevant processes, including cell proliferation, adhesion, and invasion. As an underlying mechanism, Rab31 is presumed to act as a molecular switch between a more proliferative and an invasive phenotype. This prompted us to analyze whether Rab31 overexpression in breast cancer cells affects expression of genes involved in epithelial-to-mesenchymal transition (EMT)-like processes when compared to Rab31 low-expressing cells. METHODS: Commercially available profiler PCR arrays were applied to search for differentially expressed genes in Rab31 high- and low-expressing CAMA-1 breast cancer cells. Differential expression of selected candidate genes in response to Rab31 overexpression in CAMA-1 cells was validated by independent qPCR and protein assays. RESULTS: Gene expression profiling of key genes involved in EMT, or its reciprocal process MET, identified 9 genes being significantly up- or down-regulated in Rab31 overexpressing CAMA-1 cells, with the strongest effects seen for TGFB1, encoding TGF-ß1 (> 25-fold down-regulation in Rab31 overexpressing cells). Subsequent validation analyses by qPCR revealed a strong down-regulation of TGFB1 mRNA levels in response to increased Rab31 expression not only in CAMA-1 cells, but also in another breast cancer cell line, MDA-MB-231. Using ELISA and Western blot analysis, a considerable reduction of both intracellular and secreted TGF-ß1 antigen levels was determined in Rab31 overexpressing cells compared to vector control cells. Furthermore, reduced TGF-ß activity was observed upon Rab31 overexpression in CAMA-1 cells using a sensitive TGF-ß bioassay. Finally, the relationship between Rab31 expression and the TGF-ß axis was analyzed by another profiler PCR array focusing on genes involved in TGF-ß signaling. We found 12 out of 84 mRNAs significantly reduced and 7 mRNAs significantly increased upon Rab31 overexpression. CONCLUSIONS: Our results demonstrate that Rab31 is a potent modulator of the expression of TGF-ß and other components of the TGF-ß signaling pathway in breast cancer cells.

3D Models for Ovarian Cancer.

The main reasons for the slow progress in improving survival outcomes for ovarian cancer are the 'one-size-fits-all' therapy and lack of clinically relevant experimental models that represent the advanced stages of the human disease. The interaction of tumour cells with their surrounding niche, the tumour microenvironment, influences the spread of ovarian cancer cells within the peritoneum and their responses to therapeutics. Scientists are increasingly using 3D cell culture models to dissect the role of the tumour microenvironment in cancer development and progression and the treatment of this disease. In this chapter, we will briefly describe the tumour microenvironment of ovarian cancer. Then, we will review some of the clinically relevant experimental approaches, such as spheroid, organoid and organotypic models, that have been developed for the 3D culture of ovarian cancer cells using different tools, including hydrogels, scaffolds and cancer-on-a-chip devices, to mimic selected components of the tumour microenvironment.

Kallikrein-related peptidases 4, 5, 6 and 7 regulate tumour-associated factors in serous ovarian cancer.

BACKGROUND: Tissue kallikrein-related peptidases 4, 5, 6 and 7 (KLK4-7) strongly increase the malignancy of ovarian cancer cells. Deciphering their downstream effectors, we aimed at finding new potential prognostic biomarkers and treatment targets for ovarian cancer patients. KLK4-7-transfected (OV-KLK4-7) and vector-control OV-MZ-6 (OV-VC) ovarian cancer cells were established to select differentially regulated factors. METHODS: With three independent approaches, PCR arrays, genome-wide microarray and proteome analyses, we identified 10 candidates (MSN, KRT19, COL5A2, COL1A2, BMP5, F10, KRT7, JUNB, BMP4, MMP1). To determine differential protein expression, we performed western blot analyses, immunofluorescence and immunohistochemistry for four candidates (MSN, KRT19, KRT7, JUNB) in cells, tumour xenograft and patient-derived tissues. RESULTS: We demonstrated that KLK4-7 clearly regulates expression of MSN, KRT19, KRT7 and JUNB at the mRNA and protein levels in ovarian cancer cells and tissues. Protein expression of the top-upregulated effectors, MSN and KRT19, was investigated by immunohistochemistry in patients afflicted with serous ovarian cancer and related to KLK4-7 immunoexpression. Significant positive associations were found for KRT19/KLK4, KRT19/KLK5 and MSN/KLK7. CONCLUSION: These findings imply that KLK4-7 exert key modulatory effects on other cancer-related genes and proteins in ovarian cancer. These downstream effectors of KLK4-7, MSN and KRT19 may represent important therapeutic targets in serous ovarian cancer.

Prostate Cancer-Associated Kallikrein-Related Peptidase 4 Activates Matrix Metalloproteinase-1 and Thrombospondin-1.

Prostate cancer metastasis to bone is terminal; thus, novel therapies are required to prevent end-stage disease. Kallikrein-related peptidase 4 (KLK4) is a serine protease that is overproduced in localized prostate cancer and is abundant in prostate cancer bone metastases. In vitro, KLK4 induces tumor-promoting phenotypes; however, the underlying proteolytic mechanism is undefined. The protein topography and migration analysis platform (PROTOMAP) was used for high-depth identification of KLK4 substrates secreted by prostate cancer bone metastasis-derived PC-3 cells to delineate the mechanism of KLK4 action in advanced prostate cancer. Thirty-six putative novel substrates were determined from the PROTOMAP analysis. In addition, KLK4 cleaved the established substrate, urokinase-type plasminogen activator, thus validating the approach. KLK4 activated matrix metalloproteinase-1 (MMP1), a protease that promotes prostate tumor growth and metastasis. MMP1 was produced in the tumor compartment of prostate cancer bone metastases, highlighting its accessibility to KLK4 at this site. KLK4 further liberated an N-terminal product, with purported angiogenic activity, from thrombospondin-1 (TSP1) and cleaved TSP1 in an osteoblast-derived matrix. This is the most comprehensive analysis of the proteolytic action of KLK4 in an advanced prostate cancer model to date, highlighting KLK4 as a potential multifunctional regulator of prostate cancer progression.

Lycopene's Effects on Cancer Cell Functions within Monolayer and Spheroid Cultures.

Lycopene, a compound that blocks the action of free radicals and oxygen molecules, is found in tomatoes and tomato-based products and linked to a reduced incidence of cancer. Increasing willingness of patients to maintain a healthy lifestyle by supplemental intake of nutrients and acceptance of alternative therapeutics has boosted research into nutraceuticals. The potential of lycopene to prevent or treat cancer has been investigated, but outcomes are inconsistent and its mode of action is still unknown. Further studies are needed to understand the role of lycopene in cancer prevention and treatment. The impact of lycopene on viability, proliferation, migration, and invasion of five different cancer cell lines was determined using monolayer and spheroid cultures. Cell viability was significantly reduced upon lycopene treatment at physiologically attainable concentrations. Cell proliferation, migration, and invasion did not change upon lycopene treatment. Ovarian cancer spheroids initially showed a decreased proliferation and after 14 days increased cell viability upon lycopene treatment, confirming the potential of lycopene to reduce cancer cell growth in short-term cultures and also indicate enhanced cell viability over prolonged exposure. This study cannot substantiate that lycopene inhibits cell functions associated with tumor growth, even in a 3D cancer model that mimics the natural tumor microenvironment.

A multiscale road map of cancer spheroids--incorporating experimental and mathematical modelling to understand cancer progression.

Computational models represent a highly suitable framework, not only for testing biological hypotheses and generating new ones but also for optimising experimental strategies. As one surveys the literature devoted to cancer modelling, it is obvious that immense progress has been made in applying simulation techniques to the study of cancer biology, although the full impact has yet to be realised. For example, there are excellent models to describe cancer incidence rates or factors for early disease detection, but these predictions are unable to explain the functional and molecular changes that are associated with tumour progression. In addition, it is crucial that interactions between mechanical effects, and intracellular and intercellular signalling are incorporated in order to understand cancer growth, its interaction with the extracellular microenvironment and invasion of secondary sites. There is a compelling need to tailor new, physiologically relevant in silico models that are specialised for particular types of cancer, such as ovarian cancer owing to its unique route of metastasis, which are capable of investigating anti-cancer therapies, and generating both qualitative and quantitative predictions. This Commentary will focus on how computational simulation approaches can advance our understanding of ovarian cancer progression and treatment, in particular, with the help of multicellular cancer spheroids, and thus, can inform biological hypothesis and experimental design.

Biomaterial science meets computational biology.

There is a pressing need for a predictive tool capable of revealing a holistic understanding of fundamental elements in the normal and pathological cell physiology of organoids in order to decipher the mechanoresponse of cells. Therefore, the integration of a systems bioengineering approach into a validated mathematical model is necessary to develop a new simulation tool. This tool can only be innovative by combining biomaterials science with computational biology. Systems-level and multi-scale experimental data are incorporated into a single framework, thus representing both single cells and collective cell behaviour. Such a computational platform needs to be validated in order to discover key mechano-biological factors associated with cell-cell and cell-niche interactions.

Recreating metabolic interactions of the tumour microenvironment.

Tumours are heterogeneous tissues containing diverse populations of cells and an abundant extracellular matrix (ECM). This tumour microenvironment prompts cancer cells to adapt their metabolism to survive and grow. Besides epigenetic factors, the metabolism of cancer cells is shaped by crosstalk with stromal cells and extracellular components. To date, most experimental models neglect the complexity of the tumour microenvironment and its relevance in regulating the dynamics of the metabolism in cancer. We discuss emerging strategies to model cellular and extracellular aspects of cancer metabolism. We highlight cancer models based on bioengineering, animal, and mathematical approaches to recreate cell-cell and cell-matrix interactions and patient-specific metabolism. Combining these approaches will improve our understanding of cancer metabolism and support the development of metabolism-targeting therapies.

A Self-Assembled 3D Model Demonstrates How Stiffness Educates Tumor Cell Phenotypes and Therapy Resistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense and stiff extracellular matrix (ECM) associated with tumor progression and therapy resistance. To further the understanding of how stiffening of the tumor microenvironment (TME) contributes to aggressiveness, a three-dimensional (3D) self-assembling hydrogel disease model is developed based on peptide amphiphiles (PAs, PA-E3Y) designed to tailor stiffness. The model displays nanofibrous architectures reminiscent of native TME and enables the study of the invasive behavior of PDAC cells. Enhanced tuneability of stiffness is demonstrated by interacting thermally annealed aqueous solutions of PA-E3Y (PA-E3Yh) with divalent cations to create hydrogels with mechanical properties and ultrastructure similar to native tumor ECM. It is shown that stiffening of PA-E3Yh hydrogels to levels found in PDAC induces ECM deposition, promotes epithelial-to-mesenchymal transition (EMT), enriches CD133+/CXCR4+ cancer stem cells (CSCs), and subsequently enhances drug resistance. The findings reveal how a stiff 3D environment renders PDAC cells more aggressive and therefore more faithfully recapitulates in vivo tumors.

A Tumor Microenvironment Model of Pancreatic Cancer to Elucidate Responses toward Immunotherapy.

Pancreatic cancer is a devastating malignancy with minimal treatment options. Standard-of-care therapy, including surgery and chemotherapy, is unsatisfactory, and therapies harnessing the immune system have been unsuccessful in clinical trials. Resistance to therapy and disease progression are mediated by the tumor microenvironment, which contains excessive amounts of extracellular matrix and stromal cells, acting as a barrier to drug delivery. There is a lack of preclinical pancreatic cancer models that reconstruct the extracellular, cellular, and biomechanical elements of tumor tissues to assess responses toward immunotherapy. To address this limitation and explore the effects of immunotherapy in combination with chemotherapy, a multicellular 3D cancer model using a star-shaped poly(ethylene glycol)-heparin hydrogel matrix is developed. Human pancreatic cancer cells, cancer-associated fibroblasts, and myeloid cells are grown encapsulated in hydrogels to mimic key components of tumor tissues, and cell responses toward treatment are assessed. Combining the CD11b agonist ADH-503 with anti-PD-1 immunotherapy and chemotherapy leads to a significant reduction in tumor cell viability, proliferation, metabolic activity, immunomodulation, and secretion of immunosuppressive and tumor growth-promoting cytokines.

Combined expression of KLK4, KLK5, KLK6, and KLK7 by ovarian cancer cells leads to decreased adhesion and paclitaxel-induced chemoresistance.

OBJECTIVE: Chemoresistance is a critical feature of advanced ovarian cancer with only 30% of patients surviving longer than 5 years. We have previously shown that four kallikrein-related (KLK) peptidases, KLK4, KLK5, KLK6 and KLK7 (KLK4-7), are implicated in peritoneal invasion and tumour growth, but underlying mechanisms were not identified. We also reported that KLK7 overexpression confers chemoresistance to paclitaxel, and cell survival via integrins. In this study, we further explored the functional consequenses of overexpression of all four KLKs (KLK4-7) simultaneously in the ovarian cancer cell line, OV-MZ-6, and its impact on integrin expression and signalling, cell adhesion and survival as contributors to chemoresistance and metastatic progression. METHODS: Quantitative gene and protein expression analyses, confocal microscopy, cell adhesion and chemosensitivity assays were performed. RESULTS: Expression of α5ß1/αvß3 integrins was downregulated upon combined stable KLK4-7 overexpression in OV-MZ-6 cells. Accordingly, the adhesion of these cells to vitronectin and fibronectin, the extracellular matrix binding proteins of α5ß1/αvß3 integrins and two predominant proteins of the peritoneal matrix, was decreased. KLK4-7-transfected cells were more resistant to paclitaxel (10-100 nmol/L: 38-54%), but not to carboplatin, which was associated with decreased apoptotic stimuli. However, the KLK4-7-induced paclitaxel resistance was not blocked by the MEK1/2 inhibitor, U0126. CONCLUSIONS: This study demonstrates that combined KLK4-7 expression by ovarian cancer cells promotes reduced integrin expression with consequently less cell-matrix attachment, and insensitivity to paclitaxel mediated by complex integrin and MAPK independent interactions, indicative of a malignant phenotype and disease progression suggesting a role for these KLKs in this process.

Reactivation of embryonic nodal signaling is associated with tumor progression and promotes the growth of prostate cancer cells.

BACKGROUND: Nodal is a member of the transforming growth factor ß (TGFß) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFß ligands, it modulates androgen receptor (AR) activity. METHODS: Nodal expression was investigated using immunohistochemistry of tissue microarrays and Western blots of prostate cell lines. The functional role of Nodal was examined using Matrigel and soft agar growth assays. Cross-talk between Nodal and AR signaling was assessed with luciferase reporter assays and expression of endogenous androgen regulated genes. RESULTS: Significantly increased Nodal expression was observed in cancer compared with benign prostate specimens. Nodal was only expressed by DU145 and PC3 cells. All cell lines expressed Nodal's co-receptor, Cripto-1, but lacked Lefty, a critical negative regulator of Nodal signaling. Recombinant human Nodal triggered downstream Smad2 phosphorylation in DU145 and LNCaP cells, and stable transfection of pre-pro-Nodal enhanced the growth of LNCaP cells in Matrigel and soft agar. Finally, Nodal attenuated AR signaling, reducing the activity of a PSA promoter construct in luciferase assays and down-regulating the endogenous expression of androgen regulated genes. CONCLUSIONS: An aberrant Nodal signaling pathway is re-expressed and functionally active in prostate cancer cells.

Addressing the tumour microenvironment in early drug discovery: a strategy to overcome drug resistance and identify novel targets for cancer therapy.

The tumour microenvironment (TME) comprises not only malignant and non-malignant cells, but also the extracellular matrix (ECM), secreted factors, and regulators of cellular functions. In addition to genetic alterations, changes of the biochemical/biophysical properties or cellular composition of the TME have been implicated in drug resistance. Here, we review the composition of the ECM and different elements of the TME contributing to drug resistance, including soluble factors, hypoxia, extracellular acidity, and cell adhesion properties. We discuss selected approaches for modelling the TME, current progress, and their use in low-and high-throughput assays for preclinical studies. Lastly, we summarise the status quo of advanced 3D cancer models compatible with high-throughput screening (HTS), the technical practicalities and challenges.

Modeling the Tumor Microenvironment of Ovarian Cancer: The Application of Self-Assembling Biomaterials.

Ovarian cancer (OvCa) is one of the leading causes of gynecologic malignancies. Despite treatment with surgery and chemotherapy, OvCa disseminates and recurs frequently, reducing the survival rate for patients. There is an urgent need to develop more effective treatment options for women diagnosed with OvCa. The tumor microenvironment (TME) is a key driver of disease progression, metastasis and resistance to treatment. For this reason, 3D models have been designed to represent this specific niche and allow more realistic cell behaviors compared to conventional 2D approaches. In particular, self-assembling peptides represent a promising biomaterial platform to study tumor biology. They form nanofiber networks that resemble the architecture of the extracellular matrix and can be designed to display mechanical properties and biochemical motifs representative of the TME. In this review, we highlight the properties and benefits of emerging 3D platforms used to model the ovarian TME. We also outline the challenges associated with using these 3D systems and provide suggestions for future studies and developments. We conclude that our understanding of OvCa and advances in materials science will progress the engineering of novel 3D approaches, which will enable the development of more effective therapies.

3D Collagen-Nanocellulose Matrices Model the Tumour Microenvironment of Pancreatic Cancer.

Three-dimensional (3D) cancer models are invaluable tools designed to study tumour biology and new treatments. Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest types of cancer, has been progressively explored with bioengineered 3D approaches by deconstructing elements of its tumour microenvironment. Here, we investigated the suitability of collagen-nanocellulose hydrogels to mimic the extracellular matrix of PDAC and to promote the formation of tumour spheroids and multicellular 3D cultures with stromal cells. Blending of type I collagen fibrils and cellulose nanofibres formed a matrix of controllable stiffness, which resembled the lower profile of pancreatic tumour tissues. Collagen-nanocellulose hydrogels supported the growth of tumour spheroids and multicellular 3D cultures, with increased metabolic activity and matrix stiffness. To validate our 3D cancer model, we tested the individual and combined effects of the anti-cancer compound triptolide and the chemotherapeutics gemcitabine and paclitaxel, resulting in differential cell responses. Our blended 3D matrices with tuneable mechanical properties consistently maintain the growth of PDAC cells and its cellular microenvironment and allow the screening of anti-cancer treatments.

Stromal Cells Promote Matrix Deposition, Remodelling and an Immunosuppressive Tumour Microenvironment in a 3D Model of Colon Cancer.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC develops in a complex tumour microenvironment (TME) with both mesenchymal stromal cells (MSCs) and immune infiltrate, shown to alter disease progression and treatment response. We hypothesised that an accessible, affordable model of CRC that combines multiple cell types will improve research translation to the clinic and enable the identification of novel therapeutic targets. A viable gelatine-methacrloyl-based hydrogel culture system that incorporates CRC cells with MSCs and a monocyte cell line was developed. Gels were analysed on day 10 by PCR, cytokine array, microscopy and flow cytometry. The addition of stromal cells increased transcription of matrix remodelling proteins FN1 and MMP9, induced release of tumour-promoting immune molecules MIF, Serpin E1, CXCL1, IL-8 and CXCL12 and altered cancer cell expression of immunotherapeutic targets EGFR, CD47 and PD-L1. Treatment with PD153035, an EGFR inhibitor, revealed altered CRC expression of PD-L1 but only in gels lacking MSCs. We established a viable 3D model of CRC that combined cancer cells, MSCs and monocytic cells that can be used to research the role the stroma plays in the TME, identify novel therapeutic targets and improve the transitional efficacy of therapies.

Neuroblastoma Invasion Strategies Are Regulated by the Extracellular Matrix.

Neuroblastoma is a paediatric malignancy of the developing sympathetic nervous system. About half of the patients have metastatic disease at the time of diagnosis and a survival rate of less than 50%. Our understanding of the cellular processes promoting neuroblastoma metastases will be facilitated by the development of appropriate experimental models. In this study, we aimed to explore the invasion of neuroblastoma cells and organoids from patient-derived xenografts (PDXs) grown embedded in 3D extracellular matrix (ECM) hydrogels by time-lapse microscopy and quantitative image analysis. We found that the ECM composition influenced the growth, viability and local invasion of organoids. The ECM compositions induced distinct cell behaviours, with Matrigel being the preferred substratum for local organoid invasion. Organoid invasion was cell line- and PDX-dependent. We identified six distinct phenotypes in PDX-derived organoids. In contrast, NB cell lines were more phenotypically restricted in their invasion strategies, as organoids isolated from cell line-derived xenografts displayed a broader range of phenotypes compared to clonal cell line clusters. The addition of FBS and bFGF induced more aggressive cell behaviour and a broader range of phenotypes. In contrast, the repression of the prognostic neuroblastoma marker, MYCN, resulted in less aggressive cell behaviour. The combination of PDX organoids, real-time imaging and the novel 3D culture assays developed herein will enable rapid progress in elucidating the molecular mechanisms that control neuroblastoma invasion.

Bioengineered 3D models of human pancreatic cancer recapitulate in vivo tumour biology.

Patient-derived in vivo models of human cancer have become a reality, yet their turnaround time is inadequate for clinical applications. Therefore, tailored ex vivo models that faithfully recapitulate in vivo tumour biology are urgently needed. These may especially benefit the management of pancreatic ductal adenocarcinoma (PDAC), where therapy failure has been ascribed to its high cancer stem cell (CSC) content and high density of stromal cells and extracellular matrix (ECM). To date, these features are only partially reproduced ex vivo using organoid and sphere cultures. We have now developed a more comprehensive and highly tuneable ex vivo model of PDAC based on the 3D co-assembly of peptide amphiphiles (PAs) with custom ECM components (PA-ECM). These cultures maintain patient-specific transcriptional profiles and exhibit CSC functionality, including strong in vivo tumourigenicity. User-defined modification of the system enables control over niche-dependent phenotypes such as epithelial-to-mesenchymal transition and matrix deposition. Indeed, proteomic analysis of these cultures reveals improved matrisome recapitulation compared to organoids. Most importantly, patient-specific in vivo drug responses are better reproduced in self-assembled cultures than in other models. These findings support the use of tuneable self-assembling platforms in cancer research and pave the way for future precision medicine approaches.

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma.

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.

TGFBI Production by Macrophages Contributes to an Immunosuppressive Microenvironment in Ovarian Cancer.

The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as ßig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFß and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvß3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated ß3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFß and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.