Tissue-resident Fibroblasts Research Articles

Abstract C032: The involvement of the splanchnic program at the cellular and molecular levels in pancreatic cancer associated fibroblasts

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a dense stroma primarily composed of cancer associated fibroblasts (CAFs). Our group recently performed a lineage tracing study using genetically engineered mouse models. This study demonstrated that CAFs and pancreatic tissue resident fibroblasts (TRFs) are both derived from the fetal splanchnic mesenchyme, an area of mesoderm next to the foregut endoderm. To determine whether two major subtypes of CAFs are derived from the splanchnic mesenchyme, I analyzed co-expression of the lineage tracing marker Tomato and CAF subtype marker a smooth muscle actin (aSMA). This suggests that both myofibroblasts (aSMA high) and inflammatory fibroblasts (aSMA low) are derived from the splanchnic mesenchyme. Furthermore, we found that transcription factor Gata6 is expressed in the splanchnic mesenchyme and in some CAFs. We constructed mouse genetic models where Gata6 is specifically deleted in fibroblasts. These Gata6 conditional knock out mice had increased tumor burden, suggesting that stromally-expressed Gata6 has a tumor restraining role. We derived CAF and TRF cell lines from these genetic mouse models and their littermate normal mice. My preliminary data suggest that several important fibroblast factors, including type I collagen, aSMA, and Interleukin 6, are upregulated in the Gata6 KO CAFs and TRFs relative to normal CAFs and TRFs. In summary, our study showed that two major subtypes of pancreatic CAFs are derived from the splanchnic mesenchyme, and splanchnic factors may play a role in adult pancreatic fibroblasts to regulate the expression of critical fibroblast genes. Citation Format: Tom Walter, Lu Han, Michael Ostrowski. The involvement of the splanchnic program at the cellular and molecular levels in pancreatic 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 C032.

Differential Effects of Pancreatic Cancer-Derived Extracellular Vesicles Driving a Suppressive Environment.

Pancreatic ductal adenocarcinoma (PDAC) cells display extensive crosstalk with their surrounding environment to regulate tumor growth, immune evasion, and metastasis. Recent advances have attributed many of these interactions to intercellular communication mediated by small extracellular vesicles (sEVs), involving cancer-associated fibroblasts (CAF). To explore the impact of sEVs on monocyte lineage transition as well as the expression of checkpoint receptors and activation markers, peripheral blood monocytes from healthy subjects were exposed to PDAC-derived sEVs. Additionally, to analyze the role of sEV-associated HA in immune regulation and tissue-resident fibroblasts, monocytes and pancreatic stellate cells were cultured in the presence of PDAC sEVs with or depleted of HA. Exposure of monocytes to sEVs resulted in unique phenotypic changes in HLA-DR, PD-L1, CD86 and CD64 expression, and cytokine secretion that was HA-independent except for IL-1β and MIP1β. In contrast, monocyte suppression of autologous T cell proliferation was reduced following exposure to HA-low sEVs. In addition, exposure of stellate cells to sEVs upregulated the secretion of various cytokines, including MMP-9, while removal of HA from PDAC-derived sEVs attenuated the secretion of MMP-9, demonstrating the role of sEV-associated HA in regulating expression of this pro-tumorigenic cytokine from stellate cells. This observation lends credence to the findings from the TCGA database that PDAC patients with high levels of enzymes in the HA synthesis pathway had worse survival rates compared with patients having low expression of these enzymes. PDAC-derived sEVs have an immune modulatory role affecting the activation state of monocyte subtypes. However, sEV-associated HA does not affect monocyte phenotype but alters cytokine secretion and suppression of autologous T cell proliferation and induces secretion of pro-tumorigenic factors by pancreatic stellate cells (PSC), as has been seen following the conversion of PSCs to cancer-associated fibroblasts (CAFs). Interruption of the hexosamine biosynthetic pathway, activated in PDAC producing the key substrate (UDP-GlcNAc) for HA synthesis, thus, represents a potential clinical interception strategy for PDAC patients. Findings warrant further investigations of underlying mechanisms involving larger sample cohorts.

The role of immunosuppressive myofibroblasts in the aging process and age-related diseases.

Tissue-resident fibroblasts are mesenchymal cells which control the structural integrity of the extracellular matrix (ECM). Fibroblasts possess a remarkable plasticity to allow them to adapt to the changes in the microenvironment and thus maintain tissue homeostasis. Several stresses, also those associated with the aging process, convert quiescent fibroblasts into myofibroblasts which not only display fibrogenic properties but also act as immune regulators cooperating both with tissue-resident immune cells and those immune cells recruited into affected tissues. TGF-β cytokine and reactive oxygen species (ROS) are major inducers of myofibroblast differentiation in pathological conditions either from quiescent fibroblasts or via transdifferentiation from certain other cell types, e.g., macrophages, adipocytes, pericytes, and endothelial cells. Intriguingly, TGF-β and ROS are also important signaling mediators between immunosuppressive cells, such as MDSCs, Tregs, and M2 macrophages. It seems that in pathological states, myofibroblasts are able to interact with the immunosuppressive network. There is clear evidence that a low-grade chronic inflammatory state in aging tissues is counteracted by activation of compensatory immunosuppression. Interestingly, common enhancers of the aging process, such as oxidative stress, loss of DNA integrity, and inflammatory insults, are inducers of myofibroblasts, whereas anti-aging treatments with metformin and rapamycin suppress the differentiation of myofibroblasts and thus prevent age-related tissue fibrosis. I will examine the reciprocal interactions between myofibroblasts and immunosuppressive cells within aging tissues. It seems that the differentiation of myofibroblasts with age-related harmful stresses enhances the activity of the immunosuppressive network which promotes tissue fibrosis and degeneration in elderly individuals.

The plasticity of fibroblasts: A forgotten player in the aging process.

Tissue-resident fibroblasts are mesenchymal cells which possess an impressive plasticity in their ability to modify their properties according to the requirements of the microenvironment. There are diverse subgroups of fibroblast phenotypes associated with different tissue pathological conditions, e.g., cancers, wound healing, and many fibrotic and inflammatory conditions. The heterogeneous phenotypes can be subdivided into fibrogenic and non-fibrogenic, inflammatory and immunosuppressive subtypes as well as cellular senescent subsets. A major hallmark of activated fibroblasts is that they contain different amounts of stress fibers combined with α-smooth muscle actin (α-SMA) protein, i.e., commonly this phenotype has been called the myofibroblast. Interestingly, several stresses associated with the aging process are potent inducers of myofibroblast differentiation, e.g., oxidative and endoplasmic reticulum stresses, extracellular matrix (ECM) disorders, inflammatory mediators, and telomere shortening. Accordingly, anti-aging treatments with metformin and rapamycin inhibited the differentiation of myofibroblasts in tissues. There is evidence that the senescent phenotype induced in cultured fibroblasts does not represent the phenotype of fibroblasts in aged tissues. Considering the versatile plasticity of fibroblasts as well as their frequency and structural importance in tissues, it does seem that fibroblasts are overlooked players in the aging process.

Abstract 5847: The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter to GFP-negative recipient mice. We found that minimal proportion of pancreatic CAFs were tagged with GFP. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRF progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures persisted in subsets of CAFs in both the PDAC mouse model and human patient samples. Deletion of one of the splanchnic genes, Gata6, in CAFs resulted in increased tumor burden in the pancreas, suggesting a tumor-restraining role of Gata6 in CAFs. In summary, we found that the pancreatic epithelium and bone marrow contributes to a minimal proportion of CAFs in PDAC. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs, with a potential tumor-suppressing function. This study provides genetic approaches to robustly target CAFs in vivo, and novel insights into CAF origin, heterogeneity and function in PDAC. Citation Format: Lu Han, Yongxia Wu, Kun Fang, Sean Sweeney, Ulyss Roesner, Melodie Parrish, Khushbu Patel, Tom Walter, Julia Piermattei, Anthony Trimboli, Julia Lefler, Cynthia Timmers, Xue-Zhong Yu, Victor Jin, Michael Zimmermann, Angela Mathison, Raul Urrutia, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis [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 5847.

CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice, we show that CAFs in both invasive lobular breast cancer and triple-negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo and in vitro studies reveal the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. Functional co-culture experiments show that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data suggest that CD26+ and CD26- NFs transform into distinct CAF subpopulations in mouse models of breast cancer.

Transcriptional analysis of nasal polyps fibroblasts reveals a new source of pro-inflammatory signaling in CRSwNP.

Fibroblasts and others mesenchymal cells have recently been identified as critical cells triggering tissue-specific inflammatory responses. Persistent activation of fibroblasts inflammatory program has been suggested as an underlying cause of chronic inflammation in a wide range of tissues and pathologies. Nevertheless, the role of fibroblasts in the emergence of chronic inflammation in the upper airway has not been previously addressed. We aimed to elucidate whether fibroblasts could have a role in the inflammatory response in chronic rhinosinusitis with nasal polyps (CRSwNP). We performed whole-transcriptome microarray in fibroblast cultured from CRSwNP samples and confirmed our results by qRT-PCR. We selected patients without other associated diseases in upper airway. To investigate shifts in transcriptional profile we used fibroblasts from nasal polyps and uncinate mucosae from patient with CRSwNP, and fibroblasts from uncinate mucosae from healthy subjects as controls. This study exposes activation of a pro-inflammatory and pro-fibrotic transcriptional program in nasal polyps and CRSwNP fibroblasts when compared to controls. Our Gene-set Enrichment Analysis (GSEA) pointed to common up-regulation of several pro-inflammatory pathways in patients-derived fibroblasts, along with higher mRNA expression levels of cytokines, growth factors and extracellular matrix components. Our work reveals a potential new source of inflammatory signaling in CRSwNP. Furthermore, our results suggest that deregulated inflammatory signaling in tissue-resident fibroblasts could support a Type-2 inflammatory response. Further investigations will be necessary to demonstrate the functionality of these novel results.

Abstract C049: WT1+ cancer-associated fibroblasts promote pancreatic cancer growth

Abstract Pancreatic ductal adenocarcinoma (PDA) is characterized by dense, fibro-inflammatory stroma that is established in early pathogenesis. Cancer-associated fibroblasts (CAFs) make up a significant proportion of the tumor microenvironment and represent a heterogeneous cell population that play both tumor-promoting and tumor-restricting roles. Despite their dramatic expansion in PDA pathogenesis, it is still unclear what cells in the normal pancreas give rise to these CAFs. Lineage-tracing experiments have previously identified pancreatic stellate cells, mesothelial, and tissue-resident fibroblasts as progenitors to subsets of CAFs. In this research, we firstly pulse-labeled cells expressing the mesothelial and fibroblast marker Wilms tumor 1 (WT1) in the normal pancreas of WT1CreERT;R26tdTomato mice and used an orthotopic model of PDA to trace their contribution to the PDA tumor microenvironment. We found WT1+ cells in the normal pancreas expanded to comprise a significant proportion of PDA CAFs according to immunofluorescence. Based upon single-cell RNA-sequencing (scRNA-seq), these WT1+ precursors represent an endogenous pancreatic fibroblast population similar to previously characterized universal adventitial fibroblasts (Pi16+, Dpp4+). To determine the role of WT1 in established PDA, we labeled WT1+ cells following orthotopic tumor implantation into WT1CreERT;R26tdTomato mice, and scRNA-seq analysis revealed that both inflammatory CAFs (iCAFs) and myofibroblastic CAFs (MyCAFs) express WT1. Notably, iCAFs had higher relative expression of WT1+. Additionally, we depleted WT1+ cells with diphtheria toxin from orthotopic tumors established in WT1CreERT;R26tdTomato/iDTR mice. We found depletion of WT1+ cells reduced orthotopic tumor growth, and depleted fibroblasts from the PDA tumor microenvironment. These findings implicate WT1 as a promising lineage tracing marker for a subset of pancreatic fibroblasts and demonstrates a pro-tumorigenic role of WT1+ fibroblasts in PDA. Citation Format: Allison Bischoff, Yaqing Zhang, Wei Yan, Kristee Brown, Wenting Du, Marina Pasca di Magliano. WT1+ cancer-associated fibroblasts promote pancreatic cancer growth [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C049.

Abstract C084: Oncogenic KRAS signaling drives the activation of tissue-resident fibroblasts and is required to maintain CAF heterogeneity in pancreatic cancer

Abstract The complexity of the tumor microenvironment (TME) is one of the distinguishing features of pancreatic ductal adenocarcinoma (PDA) and is responsible for patients’ poor response to therapies. The heterogeneity of cancer-associated fibroblasts (CAFs) has been correlated to the key features of the stroma that contribute to making PDA the third-leading cause of cancer-related deaths in the United States. The recent development of FDA-approved drugs against oncogenic KRAS opened a new therapeutic avenue for the treatment of tumors that have KRAS as their driver oncogene, such as PDA. However, the precise mechanisms driving the development of the TME and the contribution of KRAS to these processes have yet to be elucidated. This poses a challenge for the prediction of the effects of KRAS inhibition on established PDA tumors. By employing spatial transcriptomic technologies on various murine models recapitulating different stages of tumor initiation and progression, from acute and chronic inflammation to PanIN and overt PDA, we were able to observe distinctive changes in the activation status of pancreatic fibroblasts. These resident activated fibroblasts (RAFs) display expression of discrete markers brought upon by inflammation (inflammatory RAFs, iRAFs) or specific to oncogenic KRAS activation (myofibroblastic RAFs, myRAFs). Importantly, these RAF populations are maintained in established tumors and are identifiable in human PDA. To assess the consequences of the disruption of KRAS signaling on CAFs and RAFs, we employed a PDA mouse model that allows for the irreversible excision of KrasG12V, the FPC model (KrasFrt-LSL-G12V-Frt; p53LSL-R172H; PDX-CRE; Rosa26FlpOERT2). Through a combination of single-cell RNA sequencing (scRNA-seq), spatial transcriptomics and fluorescent in situ hybridization combined with immunofluorescence (immunoFISH) on FPC mice before and after KrasG12V deletion, we demonstrate the profound impact of Kras ablation on the TME composition. A deep remodeling of the stroma as well as significant changes in the proportion of CAF subtypes were evident. Interestingly, we observed a decrease in CAFs with a concomitant increase in RAFs. Our results suggest that the activation of oncogenic Kras in pre-neoplastic lesions drives a unique paracrine signaling that shapes the TME and is required to maintain the CAF population in PDA. Citation Format: Giuseppina Caligiuri, Jennifer S. Thalappillil, Mojdeh Shakiba, Sandeep Nadella, Juliene Hinds, Elise Courtois, William F. Flynn, Brinda Alagesan, Georgi N. Yordanov, Benjamin Kaminow, Paul Robson, Pedro A. Perez-Mancera, Jonathan Preall, Alexander Dobin, Youngku Park, David A. Tuveson. Oncogenic KRAS signaling drives the activation of tissue-resident fibroblasts and is required to maintain CAF heterogeneity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C084.

Abstract 3645: The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small portion of pancreatic CAFs were tagged with GFP. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small portion of CAFs in PDAC, and the pancreatic epithelium contributes even less. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs. This study provides approaches to robustly target CAFs in vivo, and novel insights into CAF origin and heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Wu, Melodie Parrish, Khushbu Patel, Tony Trimboli, Julia Lefler, Xuezhong Yu, Michael Zimmermann, Angela Mathison, Raul Urrutia, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3645.

Characterization of Pulmonary Fibroblast Response to Aspergillosis fumigatus Exposure and Clinical Implications on the Development of Invasive Aspergillosis

Aspergillosis fumigatus(AF) is a ubiquitous environmental mold that spreads by producing airborne conidia (spores). Playing a crucial role in the recycling of environmental carbon and nitrogen, A. fumigatus is inhaled abundantly by animals and humans alike. Unfortunately, it is also responsible for one of the worst life‐threatening pneumonias known as invasive aspergillosis with a mortality rate above 30%, even with treatment. Healthy individuals that are immunocompetent can quickly clear inhaled AF spores before their germination via immune cells. However, in immunocompromised individuals the inability to clear the fungus allows the spores to germinate releasing a plethora of proteolytic enzymes as they infiltrate the tissue resulting in severe damage to the lung. Recent studies have shown that the proper response of the immune system hinges upon complex cellular cross‐talk between resident interstitial cells and circulating immune cells. Tissue resident fibroblasts, which are responsible for the production and maintenance of the extracellular matrix (ECM), have been recently suggested to play a pivotal role in regulating immune response. We hypothesize that A. fumigatus‐induced damage to the lungs would result in activation of pulmonary fibroblasts (PFs) and aid in protective inflammatory response. Utilizing our recently developed in vivo cre‐loxP lineage tracing mouse models we have interrogated and manipulated all activated lung fibroblasts during AF infection, clearing, and reinfection. Data from our lab shows that PFs can be activated following acute inhalation of AF spores even when they are cleared from the lungs of immunocompetent mice before pathological tissue damage occurs. Furthermore, repeated AF infections which are pathologically asymptomatic result in the accumulation of activated PFs. Here we show for the first time that fibroblast activation occurs in both transient and chronic pulmonary infections revealing a role in the immunomodulatory response and subsequent development of pulmonary disease.

Abstract PR-013: The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small proportion of pancreatic CAFs were tagged with GFP, suggesting their bone marrow origin. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small proportion of CAFs in PDAC, and the pancreatic epithelium contributes even less. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs. This study provides approaches to robustly target CAFs in vivo and novel insights into CAF heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Wu, Melodie Parrish, Khushbu Patel, Xuezhong Yu, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-013.

334 Pulling the Trigger on Tissue Decline and Rejuvenation in the connective tissue - A Novel Role for JunB

Impaired stem cell functions, which gradually fail to replenish damaged and senescent cells in distinct organ parenchyma can lead to tissue atrophy and organ aging. This failure can either be due to an intrinsic dysfunction of stem/progenitor cells, the disruption of the corresponding stem cell niche, or a combination of both. Fibroblasts in the connective tissue region of many organs, such as skin, contribute to the formation of stem cell niche. Though the impact of the connective tissue resident fibroblasts on stem cell niches and organ aging is an emerging concept, the underlying mechanisms are largely unexplored.

Myofibroblast progeny in wound biology and wound healing studies.

Fibroblasts and myofibroblasts play a myriad of important roles in human tissue function, especially in wound repair and healing. Among all cells, fibroblasts are group of cells that decide the status of wound as they maintain tissue homeostasis. Currently, the increase in the deleterious effects of chronic wound and their morbidity rate has necessitated the need to understand the influence of fibroblasts and myofibroblasts, which chiefly originate locally from tissue-resident fibroblasts to address the same. Wound pathophysiology is complex, herein, we have discussed fibroblast and myofibroblast heterogeneity in skin and different organs by understanding the phenotypical and functional properties of each of its sub-populations in the process of wound healing. Recent advancements in fibroblast activation, differentiation to myofibroblasts, proliferation and migration are discussed in detail. Fibroblasts and myofibroblasts are key players in wound healing and wound remodelling, respectively, and their significance in wound repair is discussed. An increased understanding of their biology during wound healing also gives an opportunity to explore more of fibroblast and myofibroblast focused therapies to treat chronic wounds which are clinical challenges. In this regard, in the current review, we have described the different methods for isolation of primary fibroblasts and myofibroblasts from both animal models and humans, and their characterization. Additionally, we have also provided details on possible molecular targets for better understanding of prognosis, diagnosis and treatment of chronic wounds. Information will help both researchers and clinicians in providing molecular insight that enable them for effective chronic wound management. The knowledge of intimate dialogue between the fibroblast, sub-populations like, myofibroblast and their microenvironment, will serve useful in determining novel, efficient and specific therapeutic targets to treat pathological wound conditions.

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts.

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

Bone marrow-derived macrophages converted into cancer-associated fibroblast-like cells promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic reaction caused by cancer-associated fibroblasts (CAFs), which provokes treatment resistance. CAFs are newly proposed to be heterogeneous populations with different functions within the PDAC microenvironment. The most direct sources of CAFs are resident tissue fibroblasts and mesenchymal stem cells, however, the origins and functions of CAF subtypes remain unclear. Here, we established allogeneic bone marrow (BM) transplantation models using spontaneous PDAC mice, and then investigated what subtype cells derived from BM modulate the tumor microenvironment and affect the behavior of pancreatic cancer cells (PCCs). BM-derived multilineage hematopoietic cells were engrafted in recipient pancreas, and accumulated at the invasive front and central lesion of PDAC. We identified BM macrophages-derived CAFs in tumors. BM-derived macrophages treated with PCC-conditioned media expressed CAF markers. BM-derived macrophages led the local invasion of PCCs in vitro and enhanced the tumor invasive growth in vivo. Our data suggest that BM-derived cells are recruited to the pancreas during carcinogenesis and that the specific subpopulation of BM-derived macrophages partially converted into CAF-like cells, acted as leading cells, and facilitated pancreatic cancer progression. The control of the conversion of BM-derived macrophages into CAF-like cells may be a novel therapeutic strategy to suppress tumor growth.

Pharmacological Inhibition and Genetic Knockdown of BCL9 Modulate the Cellular Landscape of Cancer-Associated Fibroblasts in the Tumor-Immune Microenvironment of Colorectal Cancer.

Cancer-associated fibroblasts (CAFs) exert a key role in cancer progression and liver metastasis. They are activated in the tumor microenvironment (TME), but their prometastatic mechanisms are not defined. CAFs are abundant in colorectal cancer (CRC). However, it is not clear whether they are raised from local tissue-resident fibroblasts or pericryptal fibroblasts and distant fibroblast precursors, and whether they may stimulate metastasis-promoting communication. B-cell lymphoma 9/B-cell lymphoma 9-like (BCL9/BCL9L) is the key transcription cofactor of β-catenin. We studied the TME of CRC with single-cell sequencing and consequently found that Bcl9 depletion caused a pro-tumor effect of CAFs, while inhibition of abnormal activation of Wnt/β-catenin signal through Bcl9 depletion benefited T-cell–mediated antitumor immune responses. We also identified and evaluated four types of CAFs in CRC with liver metastasis. In summary, we demonstrate cell type landscape and transcription difference upon BCL9 suppression in CAFs, as well as how CAF affects cancer associated immune surveillance by inhibition of Wnt signaling. Targeting the Wnt signaling pathway via modulating CAF may be a potential therapeutic approach.

Fibroblasts as immune regulators in infection, inflammation and cancer.

In chronic infection, inflammation and cancer, the tissue microenvironment controls how local immune cells behave, with tissue-resident fibroblasts emerging as a key cell type in regulating activation or suppression of an immune response. Fibroblasts are heterogeneous cells, encompassing functionally distinct populations, the phenotypes of which differ according to their tissue of origin and type of inciting disease. Their immunological properties are also diverse, ranging from the maintenance of a potent inflammatory environment in chronic inflammation to promoting immunosuppression in malignancy, and encapsulating and incarcerating infectious agents within tissues. In this Review, we compare the mechanisms by which fibroblasts control local immune responses, as well as the factors regulating their inflammatory and suppressive profiles, in different tissues and pathological settings. This cross-disease perspective highlights the importance of tissue context in determining fibroblast-immune cell interactions, as well as potential therapeutic avenues to exploit this knowledge for the benefit of patients with chronic infection, inflammation and cancer.

The Role of Pro-fibrotic Myofibroblasts in Systemic Sclerosis: From Origin to Therapeutic Targeting.

Systemic sclerosis (SSc, scleroderma) is a complex connective tissue disorder characterized by multisystem clinical manifestations resulting from immune dysregulation/autoimmunity, vasculopathy, and, most notably, progressive fibrosis of the skin and internal organs. In recent years, it has been observed that the main drivers of SSc-related tissue fibrosis are myofibroblasts, a type of mesenchymal cells with both the extracellular matrix-synthesizing features of fibroblasts and the cytoskeletal characteristics of contractile smooth muscle cells. The accumulation and persistent activation of pro-fibrotic myofibroblasts during SSc development and progression result in elevated mechanical stress and reduced matrix plasticity within the affected tissues and may be ascribed to a reduced susceptibility of these cells to pro-apoptotic stimuli, as well as their increased formation from tissue-resident fibroblasts or transition from different cell types. Given the crucial role of myofibroblasts in SSc pathogenesis, finding the way to inhibit myofibroblast differentiation and accumulation by targeting their formation, function, and survival may represent an effective approach to hamper the fibrotic process or even halt or reverse established fibrosis. In this review, we discuss the role of myofibroblasts in SSc-related fibrosis, with a special focus on their cellular origin and the signaling pathways implicated in their formation and persistent activation. Furthermore, we provide an overview of potential therapeutic strategies targeting myofibroblasts that may be able to counteract fibrosis in this pathological condition.

Abstract PR001: The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small proportion of pancreatic CAFs were tagged with GFP, suggesting their bone marrow origin. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small proportion of CAFs in PDAC, and the pancreatic epithelium contributes even less. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs. This study provides approaches to robustly target CAFs in vivo and novel insights into CAF heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Xu, Sean Sweeney, Ulyss Roesner, Melodie Parrish, Khushbu Patel, Xuezhong Yu, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR001.