CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

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

Pulmonary Fibroblasts Induce Epithelial Mesenchymal Transition and Some Characteristics of Stem Cells in Non-Small Cell Lung Cancer

Background: Increasing evidence has demonstrated that stromal cells play a pivotal role to promote breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). Upon interaction with tumor cells, CAFs promote tumor progression by providing paracrine oncogenic signals mediated by activation of various pathways including developmental pathways, integrin signaling, and the MAPK pathway in tumor cells. CAFs have also been shown to promote the survival of CTCs and help them in metastasis at distant sites. Using breast cancer patient tumor datasets, we have previously identified a microRNA signature reflective of hyperactive MAPK signaling and that is significantly associated with reduced recurrence-free and overall survival. We have established 3 primary breast CAF lines, one from a Luminal A breast cancer, one from an ER-/Her2 amplified cancer, and one from a triple negative cancer, along with several primary tumor-derived dissociated tumor (DT) culture models that are tumorigenic in vivo and vary in metastatic ability. The CAFs express several hMAPK-microRNAs preferentially compared to the DTs. In addition to the paracrine interaction of stromal and tumor cells mediated by chemokines or hormones, miRNA cross talk between stromal and tumor cells can also occur. Results: To further investigate the connection between our miRNA signature and stroma, we analyzed the TCGA and METABRIC breast cancer datasets and found that the hMAPK-miRNA identifies tumors that with signficant stromal cell infiltrate. To investigate the role of specific expression of hMAPK-miRNAs in the CAFs, CM was isolated from CAFs from "aggressive" tumors, from the "indolent" tumor, and from normal human mammary fibroblasts (HMFs) and analyzed for exosome and microRNA secretion. CAFs from the aggressive tumors secrete more exosomes and more hMAPK-microRNAs into the CM than do HMFs or CAFs from the indolent tumor. Importantly, conditioned media (CM) from the "aggressive" CAFs activate MAPK and repress ER protein, mRNA and ER 3’UTR-reporter activity in ER+ MCF-7 breast cancer cells, while HMFs and "indolent" CAFs did not. Exosomes from the "aggressive" CAFs were responsible for the ER repression. To determine if the secreted miRNA differences exhibited by the CAFs could be seen in patients, serum from breast cancer patients with metastatic breast cancer and patients without metastases was analyzed for microRNA expression. Differentially expressed circulating hMAPK-miRNAs were identified in serum from metastatic breast cancer patients compared with patients without metastatses. Further analyses of the serum for CTCs and CAFs show that serum samples from metastatic patients had a significantly higher number of CTCs with CAFs compared to serum from patients without metastases. Conclusions: Collectively these data suggest that different CAF populations have distinct abilities to influence the phenotype and behavior of associated cancer cells and that CAF secreted hMAPK-miRNAs may play important roles in breast cancer progression. They further suggest that these CAF secreted miRNAs can be found in patient serum along with circulating CAFs. Citation Format: Sanket H Shah, Phil Miller, Zheng Ao, Emilio Issa, Katherine Drews Elger, Ram Datar, Marc Lippman, Dorraya El-Ashry. Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-15.

Background: Ovarian cancer (OC) is a highly treatment-resistant malignancy, often diagnosed late due to minimal symptoms and extensive metastasis. Despite platinum-based chemotherapy, recurrence and chemoresistance highlight the need for new therapies. This study focuses on cancer-associated fibroblasts (CAFs), key TME components, to explore their impact on OC progression and identify novel CAF subtypes. Methods: CAFs were isolated from OC patient tumors via enzymatic dissociation and flow cytometry. Their markers, FAP and α-SMA, were analyzed using immunofluorescence and flow cytometry. Conditioned media from CAF cultures were tested on OC cell lines (A2780, A2780-cp, SKOV3, OVCAR3) to assess proliferation and migration via cell viability and scratch assays. Cytokines in CAF-conditioned media were quantified using ELISA. Results: CAFs were identified within OC tumors, with their expression of FAP validated in high-grade serous ovarian cancer (HGSOC) samples via immunohistochemistry and flow cytometry. CAFs were successfully isolated and cultured from patient-derived OC tumors. Flow cytometry revealed that CAFs represent a minor subset of the total tumor cell population. Morphologically, CAFs exhibited plump spindle or stellate forms. CAFs derived from HGSOC displayed elevated levels of FAP and α-SMA expression compared to those derived from non-HGSOC tumors. Exposure of four OC cell lines (A2780, A2780-cp, SKOV3, OVCAR3) to CAF-conditioned media significantly promoted their growth. Scratch assays revealed enhanced motility in two OC cell lines (SKOV3 and OVCAR3) following treatment with CAF-conditioned media. Notably, the HGSOC-derived CAF subtype, W-OVCAF7, exhibited significantly greater growth- and motility-enhancing effects compared to non-HGSOC-derived CAFs. Scratch assays further confirmed that W-OVCAF7 markedly increased the migratory capacity of both SKOV3 and OVCAR3 cells, suggesting that CAF activity may vary depending on the tumor cell type. These findings underscore the pivotal role of CAFs in modulating the proliferation and migration of OC cells. Conclusion: Tumor-derived CAFs significantly enhance OC cell proliferation and migration, underscoring their critical role in the TME. This study highlights the importance of patient-derived CAFs for precise analysis of OC dynamics and lays a foundation for therapeutic strategies targeting the CAF-TME interplay. Citation Format: Ji-Young Lee, Sung Wan Kang, Min-Seo Lee, Ok-Ju Kang, Yong-Man Kim, Shin-Wha Lee. Effects of patient-derived ovarian cancer-associated fibroblasts and tumor progression and metastasis [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 2571.

BACKGROUND: Clinical introduction of tyrosine kinases inhibitors (TKIs) for ALK+ non-small cell lung cancer (NSCLC) has improved patient survival, but it did not translate into cures as acquired resistance inevitably emerges in patients with advanced and metastatic cancers. Cancer associated fibroblasts (CAFs) have emerged as the major determinant of environmental protection. While the ability of stroma to protect tumor cells from TKI is well documented in-vitro, whether the impact of stromal protection is relevant in 3D context and in-vivo remains unresolved. Thus, we hypothesized that stromal protection impacts the initial response of tumor cells to targeted therapies. METHODS: Cancer cell lines were cultured in presence of CAF conditioned media (CM) and treated with TKIs to monitor cell viability. To interrogate molecular mechanism, loss and gain of function experiments were performed using pharmacological inhibitors and recombinant human protein respectively. To address the relevance of stromal protection in-vivo, we worked with tumor xenograft mouse model. We also used mathematical modelling to understand the tumor dynamics, using inferences from xenograft mouse models to build agent-based models. RESULTS: We observed that patient derived matched normal tissue associated fibroblast (NAFs) and CAFs, both confers various level of protection to tumor cells against TKIs. Addition of rh Hepatocyte growth factor (HGF) protein mimicked the effect of CAF CM and addition of small molecule inhibitors of cMET abolished the CAF-mediated protection to tumor cells against TKIs. Our xenograft experiment showed the significant increase in stroma/tumor ratio during the initial tumor response to targeted therapy and we observed increase in tumor proliferating cells in proximity to stroma. Our math model for stroma and pre-existing resistant population showed delay in development of resistance to TKI. CONCLUSION: Fibroblast-mediated stromal protection is critically dependent on HGF-cMET axis. Dual inhibition of ALK and MET signaling abolished the fibroblast-mediated protection and increased the cell sensitivity to ALK TKIs, suggesting the crucial role of microenvironment-mediated resistance in NSCLC. To study tumor dynamics using mathematical models, we addressed that space could be key to understand the impact of stromal protection in-vivo. Our study shows that understanding the signaling changes carried by fibroblast in response to therapy may lead to potential better combination therapy. Citation Format: Bina Desai. Impact of fibroblast mediated stromal protection on evolution of resistance to targeted therapies in NSCLC [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 PO045.

<div>AbstractPurpose:<p>To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab.</p>Experimental Design:<p>Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2–based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial.</p>Results:<p>NRG1 was expressed in HER2-positive breast cancer–derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of <i>NRG1</i> from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the <i>NRG1-</i>high group.</p>Conclusions:<p>CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab.</p></div>

To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab. Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2-based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial. NRG1 was expressed in HER2-positive breast cancer-derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group. CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab.

Estrogen signaling has been extensively studied, especially in cancers that express estrogen receptor alpha (ERα). However, little is known regarding the effect of estrogen on cancer-associated fibroblasts (CAFs). Here, we explored the role of estrogen signaling of CAFs in gastric cancer (GC) progression. We investigated the phenotypic changes in CAFs upon 17β-estradiol (E2) treatment using ERα-negative/positive CAFs, and the conditioned media (CM) collected from these were compared with regard to cancer cell proliferation, migration, and invasion. A paracrine factor was found using a cytokine array and was confirmed using qRT-PCR, western blotting, and enzyme-linked immunosorbent assays. ERα-CD147-matrix metalloproteinase (MMP) axis was confirmed by knockdown experiments using specific siRNAs. We found that a subset of CAFs expressed ERα. ERα-positive CAFs were responsive to E2, inducing ERα expression in a dose-dependent manner. Although E2 did not induce the proliferation of ERα-positive CAFs, the CM from E2-bound ERα-positive CAFs significantly promoted cancer cell migration and invasion. Cytokine array revealed that CD147 was induced in ERα-positive CAFs upon E2 treatment; this was mediated via ERα. Increased CD147 upregulated MMP2 and MMP9 in CAFs, and also influenced cancer cells in a paracrine manner to increase MMPs and CD147 in cancer cells. High CD147 expression in tumor tissue was associated with a worse prognosis in GC patients. Our data suggest that estrogen signaling activation in CAFs and the byproduct CD147 are among the critical mediators between the interplay of CAFs and cancer cells to facilitate cancer progression.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a classically heterogeneous tumor for which tumor cells comprise less than 40% of the total tumor mass. To better understand mechanisms of intercellular interactions driving tumor response or resistance to chemotherapy, it is crucial to account for the complex tumor microenvironment (TME). Specifically, cancer associated fibroblasts (CAFs) have been implicated as both a tumor promoting and tumor restraining entity through disease progression and have therefore been a focus of our evolving model system. Methods: Our group created a 3-dimensional (3D), matched coculture system of patient-derived organoids (PDO), CAFs, and immune cells obtained from patients undergoing surgical resection. PDOs grown in 3D preserves the nascent tumor architecture and better replicates the disease in vitro than traditional 2D cell lines. The fully patient-matched coculture model of human PDAC allows for mechanistic interrogation into the PDAC TME by utilizing multiparameter flow cytometry, cellular sorting, and qPCR. Additionally, pharmacotyping experiments using a 5-drug regimen were used to assess chemotherapeutic resistance allowing for targeted questions exploring patient response to standard of care chemotherapy. Results: Using a combination of direct CAF-PDO coculture and PDO with CAF-conditioned media, we examined the direct (coculture) and indirect (conditioned media) impact of CAFs on PDOs. Using flow cytometry, we showed that CAF-conditioned human complete organoid media drives increased proliferation of PDOs when compared to PDOs grown in standard media. This effect was likely due to factors that are secreted from CAFs having a synergistic effect with compounds in the human complete organoid media, as this phenotype was not replicated in traditional serum free conditioned media. In direct coculture experiments of PDOs and CAFs, PDO numbers increased over time from day 2 to day 7 when compared to monoculture of PDOs, likely due to CAF presence. Lastly, to explore how CAFs may alter chemotherapeutic efficacy, PDOs were cocultured with CAFs and assessed for chemotherapeutic resistance. Pharmacotyping results showed that PDOs sorted from the PDO-CAF coculture exhibited a change in IC50 value in comparison to those PDOs that were not cocultured with CAFs. Conclusions: Here we put forth a model of PDO and CAF coculture and demonstrated its application for examining the impact of CAFs on PDAC proliferation and chemotherapeutic response. These data demonstrate the feasibility of establishing a patient-derived matched coculture model and provide the backbone for expanding this model to include representative immune cells. Citation Format: Samantha Guinn, Joseph Tandurella, Jacquelyn Zimmerman, Richard Burkhart, Elizabeth M. Jaffee. Cancer associated fibroblasts are regulators of the tumor microenvironment in human pancreatic ductal adenocarcinoma [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 C036.

Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and significantly contribute to immune evasion. We investigated the effects of CAFs on the immune function of CD4+ and CD8+ T cells in non-small cell lung cancer (NSCLC). We isolated CAFs and normal fibroblasts (NFs) from tumors and normal lung tissues of NSCLC patients, respectively. CAFs were co-cultured with activated T cells to evaluate their immune regulatory function. We investigated the effect of CAF conditioned medium (CAF-CM) on the cytotoxicity of T cells. CAFs were also co-cultured with activated peripheral blood mononuclear cells and further incubated with cyclooxygenase-2 (COX2) inhibitors to investigate the potential role of COX2 in immune evasion. CAFs and NFs were isolated from the lung tissues (n = 8) and lymph nodes (n = 3) of NSCLC patients. Immune suppressive markers, such as COX2 and PD-L1, were increased in CAFs after coculture with activated T cells. Interestingly, CAFs promoted the expression of PD-1 in CD4+ and CD8+ T cells, and strongly inhibited T cell proliferation in allogenic and autologous pairs of CAFs and T cells. CAF-CM decreased the cytotoxicity of T cells. COX2 inhibitors partially restored the proliferation of CD4+ and CD8+ T cells, and downregulated the expression of COX2, prostaglandin E synthase, prostaglandin E2, and PD-L1 in CAFs. CAFs promote immune evasion by suppressing the function of CD4+ and CD8+ T cells via their effects on COX2 and PD-L1 in NSCLC. The immunosuppressive function of CAFs could be alleviated by COX2 inhibitors.

The tumor microenvironment is composed of different types of stromal cells and a key component of tumor progression. It has been hypothesized that these stromal components are functionally organized to promote the survival of cancer cells. A specific subset of stromal cells, termed cancer-associated fibroblasts (CAFs), secretes factors that promote tumorigensis. To investigate whether CAFs constitute a specific microenvironment conductive for lung cancer progression, we performed in vitro and in vivo experiments using CAFs derived from primary lung cancer in conjunction with the human lung adenocaricinoma cell lines. We first showed that conditioned medium (CM) from human CAFs was biologically active. When human lung adenocarcinoma cell lines (A549 and NCI-H358) were cultured with CM from CAFs, epithelial cells became spindle and scattered as well as decreased cell surface E-cadherin. Further CM increased N-cadherin and vimentin expression, which is a hallmark of an epithelial-mesenchymal-transition (EMT). Coculture experiments using traswell plates with inserts of pore size 3 μm also showed EMT change in cancer cells. CM led cancer cells to enhance expression of stem cell markers such as CD44 and CD133 mRNA levels by ten times. It also triggered the acquisitions of cancer stemness such the ability of resistance to cisplatin or gefitinib as well as sphere formation. These phenotype changes associated with EMT and stemness were inhibited by the addition of TGF-β inhibitor, SB431542 (10 μM). The s.c. coinjection of human lung adenocarcinoma cell lines and human CAFs to the mice result in a high rate of tumor formation compared with the injection of these cell lines without CAFs. A decreased expression of epithelial markers and upregulation of angiogenesis and proliferative index were detected in specimens from mice with coinjection of these cells. TGF β inhibitor attenuated the tumor formation enhanced by coinjection with CAFs. The compound was injected i.p. at 0.2 mg/kg (1 per day, 5 per week for 2 weeks) after coinjection. These results indicate that the paracrine interplay between CAFs and cancer cells mediated by TGF-β signaling leads to an EMT driven gain of cancer stem cell properties and suggest that the CAFs contribute a specific niche for tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 54. doi:1538-7445.AM2012-54

Breast cancer is the most prevalent cancer among women worldwide with estrogen receptor-positive (ER+) cancer constituting 70% of all breast cancers. Although ER+ breast cancers initially respond well to ER-targeted therapies and CDK4/6 inhibitors, approximately 30% patients acquire resistance to targeted therapies. In addition to intrinsic resistance that tumors develop, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment, also contribute to treatment-resistance and poor survival. The role of secreted metabolites by CAFs has been poorly studied in the context of targeted drug resistance. Therefore, to investigate the mechanism of stroma-mediated resistance to CDK4/6-inhibitors, we propagated patient-derived organoids (PDOs) and CAFs from treatment-naïve and treatment-resistant ER+ breast tumors. Our data show that the CAF conditioned media containing the polar metabolite fraction stimulates resistance to CDK4/6 inhibitors. Therefore, to identify metabolites that drive resistance, we performed an untargeted metabolomics on CAF conditioned media. We observed that the CAF conditioned media was enriched for TCA cycle intermediates, including succinate. Succinate is an oncometabolite that has an essential function in energy metabolism. Aside from causing metabolic reprogramming, succinate can be transported out of the mitochondria to perform other biological processes, such as epigenetic modifications and pseudohypoxia. Furthermore, succinate in the extracellular space binds to the succinate receptor SUCNR1 and induces G-protein coupled receptor-mediated signaling in cells expressing SUCNR1. To assess mechanism of resistance that these metabolites might induce, we treated breast cancer cells with the most enriched metabolites we had identified in our metabolomics assay. These experiments identified succinate as driver of CDK4/6 inhibitor resistance in ER+ breast cancer cells. Furthermore, treating tumor cells with SUCNR1 antagonist increased their sensitivity to CDK4/6-inhibitors, showing that succinate mediated resistance is conveyed via SUCNR1. This suggests that CAF secreted succinate plays a role in supporting a pro-tumor environment by promoting drug resistance towards CDK4/6 inhibitors. Defining this mechanism of resistance will elucidate potential therapeutic targets to augment patient response to therapy. Citation Format: Jenny Hogstrom, Boryana Petrova, Camila Perea, Madelyn Ward, Gerburg Wulf, Laura Collins, Naama Kanarek, Taru Muranen. Mechanisms of succinate-mediated drug resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1222.

Cancer-associated fibroblasts (CAFs) within the tumor microenvironment highly contribute to cancer progression and poor prognosis. Recent findings introduce pirfenidone, a medication for pulmonary fibrosis, as a promising repurposed candidate for inhibiting CAFs. Moreover, photobiomodulation (PBM), or low-level laser therapy, has surfaced as a potential modality against cancer; however, its impact on CAFs is not thoroughly comprehended. The present study scrutinized the effects of PBM, pirfenidone, and PBM+pirfenidone on CAFs, and their consequent impact on cancer cells using the CAF-conditioned media. After treating CAFs with PBM at wavelengths 660 and 980 nm with/without PFD and then culturing cancer cells in the prepared conditioned media, cell viability, gene expression, migration, and clonogenic potential were evaluated. Results indicated that PBM reduced the anti-fibrogenic effects of pirfenidone on CAFs and increased their viability. PBM compensated for inhibited migration-inducing effects of CAFs by pirfenidone on cancer cells and promoted their clonogenic potential. Gene expression studies uncovered modulation of EMT and stemness-related genes by PBM and pirfenidone. In conclusion, findings propose that PBM could maintain pro-tumorigenic effects of CAFs even following treatment with the anti-fibrotic agent pirfenidone; therefore, PBM should cautiously be used against cancer before further investigations expound the underlying mechanisms and optimize treatment protocols.

Simple SummaryCancer-associated fibroblasts (CAFs) represent an important stromal cell population of pancreatic cancer in which multiple CAF subtypes have been identified. CAFs engage in a bidirectional crosstalk with cancer cells, continuously adapting their metabolism to external factors, such as chemotherapy. In this review, we summarize recently identified CAF subtypes in pancreatic cancer and discuss how CAFs shape cancer cell metabolism through several mechanisms, notably metabolite exchange, paracrine signaling, desmoplasia/hypoxia and acidosis.Despite extensive research, the 5-year survival rate of pancreatic cancer (PDAC) patients remains at only 9%. Patients often show poor treatment response, due partly to a highly complex tumor microenvironment (TME). Cancer-associated fibroblast (CAF) heterogeneity is characteristic of the pancreatic TME, where several CAF subpopulations have been identified, such as myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen presenting CAFs (apCAFs). In PDAC, cancer cells continuously adapt their metabolism (metabolic switch) to environmental changes in pH, oxygenation, and nutrient availability. Recent advances show that these environmental alterations are all heavily driven by stromal CAFs. CAFs and cancer cells exchange cytokines and metabolites, engaging in a tight bidirectional crosstalk, which promotes tumor aggressiveness and allows constant adaptation to external stress, such as chemotherapy. In this review, we summarize CAF diversity and CAF-mediated metabolic rewiring, in a PDAC-specific context. First, we recapitulate the most recently identified CAF subtypes, focusing on the cell of origin, activation mechanism, species-dependent markers, and functions. Next, we describe in detail the metabolic crosstalk between CAFs and tumor cells. Additionally, we elucidate how CAF-driven paracrine signaling, desmoplasia, and acidosis orchestrate cancer cell metabolism. Finally, we highlight how the CAF/cancer cell crosstalk could pave the way for new therapeutic strategies.

BackgroundBreast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.MethodsWe utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.ResultsWe found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.ConclusionWe found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.