Targeting lipid metabolism in CAFs: A therapeutic opportunity in solid tumors.

BackgroundOsteosarcoma (OS), with a peak incidence during the adolescent growth spurt, is correlated with poor prognosis for its high malignancy. The tumor microenvironment (TME) is highly complicated, with frequent interactions between tumor and stromal cells. The cancer-associated fibroblasts (CAFs) in the TME have been considered to actively involve in the progression, metastasis, and drug resistance of OS. This study aimed to characterize cellular heterogeneity and molecular characterization in CAFs subtypes and explore the potential targeting therapeutic strategies to improve the prognosis of OS patients. MethodsThe single-cell atlas of human OS tumor lesions were constructed from the GEO database. Then significant marker genes and potential biological functions for each CAFs subtype were identified and explored using the Seurat R package. Next, by performing the survival analyses and constructing the risk scores for CAFs subtypes, we aimed to identify and characterize the prognostic values of specific marker genes and different CAFs subtypes. Furthermore, we explored the therapeutic targets and innovative drugs targeting different CAFs subtypes based on the GDSC database. Finally, prognoses related CAFs subtypes were further validated through immunohistochemistry (IHC) on clinical OS specimens. ResultsOverall, nine main cell clusters and five subtypes of CAFs were identified. The differentially expressed marker genes for each CAFs clusters were then identified. Moreover, through Gene Ontology (GO) enrichment analysis, we defined the CAFs_2 (upregulated CXCL14 and C3), which was closely related to leukocyte migration and chemotaxis, as inflammatory CAFs (iCAFs). Likewise, we defined the CAFs_4 (upregulated CD74, HLA-DRA and HLA-DRB1), which was closely related to antigen process and presentation, as antigen-presenting CAFs (apCAFs). Furthermore, Kaplan-Meier analyses showed that CAFs_2 and CAFs_4 were correlated with poor clinical prognosis of OS patients. Meanwhile, therapeutic drugs targeting CAFs_2 and CAFs_4, such as 17-AAG/Docetaxel/Bleomycin and PHA-793887/NG-25/KIN001-102, were also explored, respectively. Finally, IHC assay confirmed the abundant CAFs_2 and CAFs_4 subtypes infiltration in the OS microenvironment compared with adjacent tissues. ConclusionOur study revealed the diversity, complexity, and heterogeneity of CAFs in OS, and complemented the single-cell atlas in OS TME.

The tumor microenvironment (TME) is a critical driver of cancer progression, metastasis, and therapeutic resistance. Among its various cellular components, cancer associated fibroblasts (CAFs) are particularly essential due to their ability to remodel the extracellular matrix (ECM), secrete growth factors and cytokines, and interact with other cell types to promote tumorigenesis. The heterogeneity of CAFs, increasingly recognized as subtypes including myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs), adds complexity to their role in cancer biology. This diversity also complicates the therapeutic potential, yet targeting CAFs has emerged as a promising strategy to overcome the therapeutic resistance associated with the TME. However, the development of effective therapies is challenging due to the complex interactions between CAF subtypes, immune cells, and tumor cells. This dissertation addresses these challenges by exploring innovative approaches to selectively target different CAF subtypes, aiming to disrupt their pro-tumorigenic functions and improve the efficacy of chemotherapy. The first part of this dissertation discusses the biology of CAFs as well as potential therapeutic strategies. Special attention is given to pancreatic ductal adenocarcinoma (PDAC), one of the most desmoplastic tumors, underscoring the importance of developing biologically relevant models that reflect the TME complexity. The second part of this dissertation introduces a polymeric nanofiber system designed for the controlled co-delivery of a p21-activated kinase 1 inhibitor, FRAX597, and the chemotherapeutic agent paclitaxel. This system was tested in stroma-rich breast cancer models highlighting the potential of targeting CAFs to overcome chemoresistance in solid tumors. In the third part, integrins α5 and α11 were identified as potential drug delivery targets for iCAFs/myCAFs and myCAFs only, respectively. Utilizing novel peptides AV3 and AXI, surface-functionalized polymeric nanoparticles showed selective targeting and enhanced PDAC biodistribution. Building on this, the fourth part investigates the therapeutic efficacy of these nanoparticles in delivering a monoacylglycerol lipase inhibitor, URB602 and an aryl hydrocarbon receptor agonist, YL109 to CAF subtypes, resulting in TME modulation in preclinical KPC model. Finally, the fifth part explores the potential of specific targets inhibition within CAFs, including fibroblast growth factor receptor-4 and dipeptidyl peptidase-4, demonstrating therapeutic suppression of iCAFs, myCAFs and significant modulation of PDAC TME.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is resistant to many available therapies including immunotherapy because of its highly complex tumor microenvironment (TME). PDAC TME consists of a significant proportion of stromal cells,...

Introduction: The tumor microenvironment (TME) is critical in determining tumor development and progression. Among stromal cells in TME, cancer associated fibroblasts (CAFs) play a central role and are involved in various signals through their interaction with cancer cells in most solid tumors, leading to cancer progression and acquisition of drug resistance. Under these TME conditions, tumor cell death such as necrosis occurs, which in turn, results in the ectopic release of bioactive molecules collectively termed as damage-associated molecular patterns (DAMPs), which has been reported to play an important role in cancer progression. The purpose of this study is to clarify the effects of cancer cell-derived HMGB1, a representative DAMPs, on CAFs and to investigate whether it could be a potential therapeutic target. Methods: We established CAFs from resected non-small cell lung cancer (NSCLC) specimens and investigated the biological activity of HMGB1 on CAFs using in vitro co-culture models with NSCLC cell lines. Furthermore, we assessed the therapeutic efficacy of targeting cancer cell-derived HMGB1 using anti-HMGB1 neutralizing antibody. Results: Intracellular HMGB1 was expressed more in NSCLC cells than in normal bronchial epithelial cells and CAFs, and released from cancer cells extracellularly upon treatment with anticancer drugs. The stimulation with recombinant HMGB1 promoted the proliferation of CAFs in a dose-dependent manner, but not lung cancer cells. Co-culture with CAFs promoted cancer cells’ migratory ability and drug resistance, and stimulation with recombinant HMGB1 further enhanced these promoting effects, while administration of anti-HMGB1 neutralizing antibody suppressed these effects. On the other hand, these inhibitory effects were not observed in the absence of CAFs, suggestingHMGB1 has effect on CAFs, but not direct on cancer cells. Conclusion: HMGB1 plays an important role in cancer progression and acquisition of therapeutic resistance by upregulating the CAFs’ proliferation and activity and could be a therapeutic target in NSCLC. Citation Format: Tomohiro Habu, Keiichi Date, Ken Suzawa, Mao Yoshikawa, Masayoshi Ohki, Kazuma Iwata, Naoki Matsuda, Yin Min Thu, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. The role of high mobility group box-1 protein (HMGB1) in the microenvironment of non-small cell lung cancer [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 2502.

Background/Aim: Tumor microenvironment plays supportive roles for cancer cells of proliferating, invading and spreading systemically. Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) are deemed to be essential constituents forming cancer-surrounding niche. However, the cross-talks between CAFs and TAMs in the settings of hepatocellular carcinoma (HCC) has yet to be deciphered. The lack of a reliable system simulating in vivo-microenvironment has been one of the obstacles hampering investigation of CAFs and TAMs. By using the newly-established model consisting of fibroblasts directly recovered from the disease liver, we aimed to clarify the mechanisms of CAFs modulating the macrophage differentiation and malignant potential of cancer cells in order to search for therapeutic targets. Methods: We enrolled three patients who underwent the resection of HCC and two who did living donor liver transplantation. From the resected or the explanted livers, fibroblasts from cancer and its adjacent tissues were separated by tissue digestion. We examined their ontogeny by the expression of fibroblast-specific markers. For the functional analyses, we examined invasiveness of HuH7 cells on matrigel and migratory response of monocytes in the presence of conditioned media (CM) from the fibroblasts. We assessed the phenotypes/function of macrophages after the differentiation from monocytes in the presence of M-CSF and the CM. We comprehensively assayed cytokine/chemokine in the CM to identify functionally-relevant factors. Results): After the culture, we established three types of fibroblasts, CAFs, cirrhotic liver fibroblasts (LCFs) and non-cancerous fibroblasts (NFs). All of these were positive for vimentin, SMA, PDGFRa and FAP (>99%), confirming that they are ontologically fibroblasts instead of being HCC, hepatocytes or biliary cells. The invasiveness of HuH7 were higher in the presence of the CAF- or LCF-CM compared to those with NF-CM. Similar superiority was observed with the CAF- or LCF-CM in the migratory ability of monocytes. In the process of macrophage differentiation, the CAF- or LCF-CM were capable of polarizing macrophages into TAM/M2 subtypes, as evidenced by their higher production of IL-10 but lesser IL-12. In the CAF- or LCM-CM, the levels of IL6, IL-8, CCL2 and GRO were higher than those in the NF-CM. Conclusion: The CAFs as well as LCFs directly recovered from the cirrhotic liver are functionally competent of driving TAM/M2 differentiation and providing malignant potential to cancer cells, thereby tuning the microenvironment favoring HCC development. Citation Format: Yohei Mano, Tatsuya Kanto, Hirotaka Shoji, Schiyo Yoshio, Masaya Sugiyama, Yosuke Osawa, Kiminori Kimura, Ken Shirabe, Yoshihiko Maehara, Masashi Mizokami. Cancer-associated or cirrhosis fibroblasts recovered from hepatocellular carcinoma promote macrophages and cancer cells to progressive phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3354. doi:10.1158/1538-7445.AM2015-3354

Tumor microenvironment (TME) has been demonstrated to play a significant role in tumor initiation, progression, and metastasis. Cancer-associated fibroblasts (CAFs) are the major component of TME and exhibit heterogeneous properties in their communication with tumor cells. This heterogeneity of CAFs can be attributed to various origins, including quiescent fibroblasts, mesenchymal stem cells (MSCs), adipocytes, pericytes, endothelial cells, and mesothelial cells. Moreover, single-cell RNA sequencing has identified diverse phenotypes of CAFs, with myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) being the most acknowledged, alongside newly discovered subtypes like antigen-presenting CAFs (apCAFs). Due to these heterogeneities, CAFs exert multiple functions in tumorigenesis, cancer stemness, angiogenesis, immunosuppression, metabolism, and metastasis. As a result, targeted therapies aimed at the TME, particularly focusing on CAFs, are rapidly developing, fueling the promising future of advanced tumor-targeted therapy.

Development and progression of cancer are often facilitated by deregulation of gene expression and accumulation of somatic mutations. Therefore, major research efforts have focused on cancer cells to identify these disease-related genes and mutations, and investigators have developed therapies that target such deregulated genes and mutations and used them for a variety of cancers. In addition to cancer cells, the complex and relatively unexplored tumor microenvironment plays an important role in cancer progression. However, therapeutic regimens targeting tumor stromal cells, particularly cancer-associated fibroblasts (CAFs), have yet to be developed. Because the tumor-supportive roles of CAFs are increasingly being recognized, researchers have begun to evaluate the potential of inhibiting tumor progression via stromal ablation. However, multiple studies demonstrated that ablation of activated myofibroblasts in tumors actually promotes tumor progression and reduces survival.1,2 Although inhibiting tumor progression by targeting stroma via global depletion of CAFs may not be viable, reprogramming CAFs by targeting deregulated genes presents the opportunity for developing novel cancer therapy. CAF reprogramming allows silencing of CAF-derived tumor-promoting factors while preserving the overall structural integrity of the extracellular matrix. We believe that reprogramming CAFs in the tumor microenvironment can inhibit tumor progression, thereby presenting an alternative approach to the treatment of cancer. Transcriptome profiling of laser-microdissected ovarian tumor samples enabled us to identify stroma-exclusive genes that are deregulated in tumors. This cannot be achieved using bulk tumor samples containing various amounts of epithelial and stromal components. Using laser-microdissected tissue samples, we identified a CAF gene expression signature for high-grade serous ovarian cancer (HGSOC).3 Immunolocalization of microfibrillar-associated protein 5 (MFAP5) in tissues samples obtained from HGSOC patients and cell culture studies demonstrated that whereas MFAP5 was expressed by cancer cells in a small group of patients, CAFs had markedly higher MFAP5 expression than that in cancer cells, and MFAP5 was exclusively expressed by CAFs in the majority of HGSOC patients.4,5 We further evaluated the functional roles of MFAP5 in ovarian cancer progression. We observed that MFAP5 was actively secreted by CAFs into the extracellular matrix, where MFAP5 binds to the αvβ3 integrin receptors on the surface of ovarian cancer cells. Engagement of αvβ3 integrin with MFAP5 on cancer cells subsequently activates the calcium-dependent focal adhesion kinase/extracellular signal-regulated kinase/cAMP response element-binding protein signaling pathway, which leads to upregulation of troponin C type 1 (TNNC1) expression. TNNC1 facilitates the formation and rearrangement of the F-actin cytoskeleton and enhances the motility of ovarian cancer cells via induction of traction force.5 TNNC1 is commonly expressed by muscle cells, including those in skeletal and cardiac muscles, and is involved in the contraction of these muscles. Further survival analysis demonstrated that TNNC1 is an independent biomarker for poor prognosis for HGSOC. Besides stromal-epithelial cross-talk mediated by CAF-derived MFAP5, we found a positive correlation between MFAP5 expression and microvessel density,4,5 suggesting that CAF-derived MFAP5 modulates tumor progression via increased angiogenesis. However, the underlying molecular mechanism of how MFAP5 stimulates angiogenesis remains to be elucidated. Taken together with the fact that high stromal MFAP5 expression is closely associated with poor outcome of HGSOC and functional studies demonstrated the stimulatory effects of MFAP5 on the motility and invasive potential of ovarian cancer cells, reprogramming of CAFs by targeting MFAP5 may be a novel therapeutic strategy for ovarian cancer that may improve survival in HGSOC patients. To determine the effect of reprogramming of CAFs by targeting MFAP5 on ovarian cancer progression, we injected murine MFAP5-targeting small interfering RNA (siRNA) incorporated into chitosan nanoparticles into ovarian cancer-bearing mice. We observed that this treatment successfully silenced MFAP5 expression in CAFs, decreased the expression of MFAP5's downstream target gene TNNC1 in tumor cells, and inhibited ovarian tumor growth, invasion, and metastasis.5 These findings suggested that delivery of siRNAs specific to CAF-expressing genes could reprogram CAFs and lead to inhibition of tumor progression (Fig. 1). CAF reprogramming can be further improved with targeted delivery of siRNA sequences using vehicles that specifically target CAFs. This improved specificity can certainly increase treatment efficacy, reduce treatment dosages required for gene silencing, and possibly reduce side effects. A number of such targeted delivery platforms are being developed and evaluated.6 Figure 1. CAF reprogramming using MFAP5-tareting siRNA delivered by chitosan nanoparticles significantly reduces the level of CAF-derived MFAP5 protein in the tumor microenvironment. Shutting down MFAP5-modulated activation of αvβ3 integrin and ... In summary, CAF-derived MFAP5 plays important roles in modulating cancer progression and has proven to be a potential therapeutic target for ovarian cancer. These findings suggest that CAF reprogramming via targeted gene silencing in these cells is a new paradigm for ovarian cancer treatment. Also, it may synergize with other treatment regimens that target cancer cells to increase treatment efficacy.

It is now well appreciated that tumor growth depends on the tumor microenvironment (TME) comprising various non-malignant stromal cell types, including cancer-associated fibroblasts (CAFs). Cancer-associated fibroblasts (CAFs) are responsible for secreting the extracellular matrix (ECM) prominent in certain solid tumors such as pancreatic adenocarcinoma (PDAC). Despite our increasing knowledge of CAFs, therapeutic targeting of CAFs is currently not a feasible strategy, as CAFs are highly heterogeneous and extremely plastic, with each CAF population able to adopt both tumor-promoting and -restraining functions. We hypothesize that the plasticity and functional heterogeneity of CAFs can be explained by their flexible metabolism in response to environmental cues and the existence of discrete metabolic states within CAF subpopulations. While metabolic states can be predicted from single-cell transcriptomic data, this does not provide information on metabolic activity and potential, nor does it allow perturbations required for validation. Scalable and tractable methods to assess metabolic activity and CAF heterogeneity in parallel at the single cell level are needed to distinguish numerous existing CAF states do not exist. To address this need, in this work, we developed a strategy to study CAF metabolic activity and phenotypic heterogeneity at the single-cell level. We designed and optimized a 25-plex immunophenotyping panel of CAF and fibroblast markers that can be combined with numerous metabolic probes via Full Spectrum Profiling (FSP) flow cytometry. In addition, we compiled an analysis pipeline to facilitate the analysis of high-dimensional flow cytometry data. Using established 2D and 3D mono- and co-culture assays, we demonstrate the potential of our panel to be multiplexed with chemical probes for cell proliferation, lipid content, lipid uptake, fatty acid oxidation, glucose uptake, cholesterol content, hypoxia, or mitochondrial potential. Deploying our strategy in a KPC organoid-derived murine model of PDAC, we observed subpopulations of CAFs with differing metabolic potentials that are consistent with our predictions from transcriptomic metabolic modeling. With the high throughput and single-cell nature of FSP, our strategy represents a novel and modular tool for studying CAF phenotypic and metabolic heterogeneity. Citation Format: Kevin Muñoz Forti, Marta Storl-Desmond, Daniel Martínez, Sayana Issac, Shan Cao, Simon Schwörer. Full spectrum profiling flow cytometry to study cancer-associated fibroblast heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2667.

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for α-smooth muscle actin and several other contractile proteins; 2) ‘inflammatory’ CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment (TME) and are known for their role in supporting tumor growth and progression through interactions with cancer cells and other stromal elements. Liver metastatic CAFs (lmCAFs), in particular, play an important role in establishing the secondary niche that facilitates tumor metastasis. Reprogramming the stroma, particularly targeting the pro-tumorigenic functions of lmCAFs, has emerged as a promising therapeutic intervention to disrupt the supportive environment tumors rely on for progression. An initial drug screen of FDA-approved oncology therapies was conducted on patient-derived colorectal cancer (CRC) CAFs and tumor organoids (PDTOs) to identify compounds that may be re-purposed as stroma targeting agents. All-trans retinoic acid (ATRA) was shown to specifically inhibit the proliferation of CAFs without affecting CRC-PDTOs. ATRA is an active metabolite of Vitamin A that exerts its canonical biological effects by binding to nuclear retinoic acid receptors (RAR). The differential RAR expression in multiple CAFs and PDTOs was confirmed with bulk RNA sequencing, with higher expression found in the CAF lines. Subsequent analysis included the qPCR assessment of glycolysis enzyme levels, CAF activation markers, and ATRA signaling in lmCAFs following treatment with ATRA for 5 days. Protein expression of key components of the TGF-b signaling pathway, including SMAD 2/3, Akt, and b-catenin, were also analyzed. Treatment of lmCAFs with ATRA induced expression of RARb and ALDH1A1, biomarkers associated with ATRA signaling pathways. In one patient-derived CAF line, ATRA treatment exhibited a decrease in CAF activation markers (e.g. aSMA) and glycolytic enzymes (e.g. lactate dehydrogenase). On the other hand, another CAF line showed increased fibroblast activation and a shift away from metabolic dependency on glycolysis upon ATRA treatment. Additionally, ATRA targeted TGF-b signaling through the pSMAD 2/3 and Akt pathways, highlighting the multifaceted impact on CAF function and heterogeneity. ATRA effectively reprograms some CAFs by inhibiting their proliferation and altering key signaling pathways, thereby disrupting the pro-tumorigenic environment of the TME. The results of this study suggest that ATRA holds potential as a therapeutic strategy to target and reprogram the CAFs within the permissive stromal components essential for tumor progression. Citation Format: Brandon Choi, Yuyuan Zhou, Seungil Kim, Shannon M. Mumenthaler. Targeting colorectal cancer-associated fibroblasts with all-trans retinoic acid to reprogram the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C031.

Cancer-associated fibroblasts (CAFs) are key components of the dense, proliferating stroma observed in pancreatic ductal adenocarcinoma (PDAC), and CAF subpopulations drive tumor heterogeneity and play a major role in PDAC progression and drug resistance. CAFs consist of heterogenous subpopulations such as myoblastic CAF (myCAF) and inflammatory CAF (iCAF), and each has distinct essential roles. However, it is not clear how CAF subpopulations are formed in PDAC. Adipose-derived MSCs (AD-MSCs), which possess a high multilineage potential and self-renewal capacity, are reported to be one of the invivo CAF sources. Here, we aimed to investigate whether AD-MSCs can act as precursors for CAFs invitro. We recorded morphological features and collected omics data from two invitro co-culture models for recapitulating clinical PDAC. Additionally, we tested the advantages of the co-culture model in terms of accurately modeling morphology and CAF heterogeneity. We showed that AD-MSCs differentiate into two distinct CAF subpopulations: Direct contact co-culture with PDAC cell line Capan-1 induced differentiation into myCAFs and iCAFs, while indirect co-culture induced differentiation into only iCAFs. Using these co-culture systems, we also identified novel CAF markers that may be helpful for elucidating the mechanisms of CAFs in the tumor microenvironment (TME). In conclusion, AD-MSCs can differentiate into distinct CAF subtypes depending on the different co-culture conditions invitro, and the identification of potential CAF markers may aid in future investigations of the mechanisms underlying the role of CAFs in the TME.

Background: Cancer associated fibroblasts (CAFs) are activated fibroblasts and an important player in the tumor microenvironment. Their activity promotes cancer cell proliferation, migration, and invasion, and metastasis. The most prognostic factor in tumor progression is metastasis. Cancer metastasis is a multi-step process that tumor cells detach from primary site, survive in the bloodstream, and seed in target organ. Although previous studies have focused on the interaction between CAFs and cancer cells in primary tumor site, the roles of CAFs in blood circulation remain largely unknown. We investigated the effect of CAFs coexisting with cancer cells in bloodstream on tumor metastasis in vivo mouse model, and also examined the effect of CAFs in subcutaneous tumor. Methods: We used female BALB/c-nu/nu mice and BALB/c mice in the experiments. Cancer cells were mouse mammary carcinoma cell lines 4T1 transfected with luciferase, colon cancer cell lines Colon 26 transfected with luciferase. Fibroblast cell lines were mouse embryonic fibroblast MEF and NIH-3T3. In venous injection mouse model, we injected 1 × 106 cancer cells alone or the same number of cancer and fibroblast co-cultured together. When we injected cancer cells transfected with luciferase, the mice were subjected to in vivo imaging system (IVIS) and measured luminescence intensity of metastatic sites. We have also harvested lung and compared its weight and metastatic nodule under microscopy. Furthermore, in subcutaneous tumor metastatic mouse model, cancer cells alone or mixed with cancer cells and CAFs were subcutaneously inoculated into the mice. Results: In the group of mice injected with cancer cells and fibroblasts, luminescent intensity of each lungs were higher than cancer cell alone. Harvested lung weight and the number of metastatic nodules were also higher in the group with cancer cells and fibroblasts.In subcutaneous model, mice inoculated with cancer cells and fibroblast had much more metastatic sites than cancer cells alone. Conclusions: Our data indicate that CAFs promote tumor metastasis by stimulating cancer cells in blood circulation, as well as in primary tumor site.These findings suggest that CAFs in both bloodstream and primary site could be a promising therapeutic target. CAF-targeted therapy could reduce tumor metastasis and improve the prognosis of cancer patients. Citation Format: Hajime Kashima, Kazuhiro Noma, Hiroaki Sato, Yuki Katsura, Takuya Kato, Toshiaki Ohara, Hiroshi Tazawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Cancer associated fibroblasts promote tumor metastasis coexisting with cancer cells in blood circulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5905. doi:10.1158/1538-7445.AM2017-5905

Cancer progression and treatment failure are driven not only by tumor-intrinsic alterations but also by dynamic interactions within the tumor microenvironment (TME). Epithelial-mesenchymal transition (EMT) and cancer-associated fibroblasts (CAFs) represent two interlinked mechanisms that promote tumor invasion, metastatic dissemination, stemness, immune evasion, and resistance to therapy. EMT is regulated by transcription factors, such as Snail, ZEB, and Twist, and by signaling pathways including TGF-β, Wnt/β-catenin, and Notch, enabling cancer cells to adopt hybrid epithelial-mesenchymal states that confer phenotypic plasticity and drug tolerance. CAFs, derived from multiple cellular sources, further reinforce EMT programs through paracrine signaling, extracellular matrix remodeling, and metabolic reprogramming. This review critically synthesizes current evidence on EMT-CAF crosstalk in oncogenesis and therapeutic resistance, highlighting emerging clinical strategies, translational challenges, and lessons from failed or limited therapeutic approaches. By emphasizing EMT plasticity and CAF heterogeneity as convergent drivers of tumor adaptability, this work provides a refined framework for developing rational combination therapies targeting both cancer cells and their supportive stroma.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains challenging to treat in part due to the dense desmoplastic stroma characteristic of the tumor microenvironment (TME). Often, less than 50% of the tumor mass is composed of malignant ductal epithelial cells. The relationship between the TME and the ductal tumor is important in tumorigenesis, metastasis, and treatment resistance. Cancer associated fibroblasts (CAFs) are an abundant cell type in the TME and have a dynamic role in both promoting and inhibiting tumor progression. Efforts to target fibroblast function therapeutically have yielded mixed results. While pre-clinical data have generated some optimism that CAF inhibition may improve clinical outcomes, this has not translated to the clinical setting. One likely reason for these inconsistent findings is the evolving understanding of CAF heterogeneity. Expanding our understanding of the interactions between the epithelial tumor and CAFs may identify new modalities for modulating the TME to augment treatment efficacy and improve patient outcomes. This requires the generation of human model systems to better interrogate intercellular interactions. Methods: We have developed an ex vivo model system to investigate interactions between ductal epithelial tumor cells and CAFs using matched patient-derived organoids (PDOs) and CAFs extracted following dissociation of surgical specimens. Flow cytometry was used to evaluate the viability of both cellular components as well as associated proliferation and cell identity. Moreover, we have used immunohistochemistry to further characterize this system while preserving the spatial relationship of the cell populations. Most recently, we employed the MULTI-seq multiplex single cell RNA sequencing (scRNA-seq) pipeline to explore the transcriptional dynamics of this system at intervals over 96 hours by examining 4 conditions with 1,500 cells sequenced per condition per time point. Results: Flow cytometry demonstrated that both PDOs and CAFs remain viable in this co-culture construct and that both cell types can be reliably identified using epithelial markers to identify organoid cells and an exclusion panel to identify the CAFs. We have further validated these findings using immunohistochemistry and demonstrated that in co-culture both PDOs and CAFs remain viable and continue to proliferate. We have also demonstrated successful extraction of both cell types from the Matrigel scaffold in which they are grown for use in downstream high dimensional genomics assays. Conclusions: We have previously demonstrated the utility of a PDO model to explore clinically relevant biological mechanisms in PDAC. However, the addition of CAFs to this model provides a more comprehensive representation of the in vivo tumor. This ex vivo system is viable and flexible for multiple end point assays and can be used to explore relevant cellular interactions and mechanisms. Our ongoing investigation will specifically focus on PDO-CAF interactions and transcriptional dynamics over time as we interrogate the scRNA-seq data. Citation Format: Jacquelyn W. Zimmerman, Genevieve Stein-O'Brien, Richard A. Burkhart, Elana J. Fertig, Elizabeth M. Jaffee. Patient-derived organoids and cancer associated fibroblasts as a co-culture model to explore cell type interactions in pancreatic cancer [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 PO-080.

Thyroid cancer is the most common endocrine malignancy. Although most thyroid cancer patients are successfully treated and have an excellent prognosis, a percentage of these patients will develop aggressive disease and, eventually, progress to anaplastic thyroid cancer. Since most patients with this type of aggressive thyroid carcinoma will die from the disease, new treatment strategies are urgently needed. Tumor cells live in a complex and dynamic tumor microenvironment composed of different types of stromal cells. Cancer-associated fibroblasts (CAFs) are one of the most important cell components in the tumor microenvironment of most solid tumors, including thyroid cancer. CAFs originate mainly from mesenchymal cells and resident fibroblasts that are activated and reprogrammed in response to paracrine factors and cytokines produced and released by tumor cells. Upon reprogramming, which is distinguished by the expression of different marker proteins, CAFs synthesize and secret soluble factors. The secretome of CAFs directly impacts different functions of tumor cells. This bi-directional interplay between CAFs and tumor cells within the tumor microenvironment ends up fostering tumor cancer progression. CAFs are therefore key regulators of tumor progression and represent an under-explored therapeutic target in thyroid cancer.