Cells And Cancer-associated Fibroblasts Research Articles

Targeting CXCR4/CXCL12 axis via [177Lu]Lu-DOTAGA.(SA.FAPi)2 with CXCR4 antagonist in triple-negative breast cancer.

Radiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role inorchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC). Public database was first interrogated to reveal the correlation between CAFs' scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen. CAFs' scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.

Fibroblast regulates angiogenesis in assembled oral cancer organoid: A possible role of NNMT.

Tumour angiogenesis is affected by various cell types in the tumour microenvironment (TME), including cancer cells and cancer-associated fibroblasts (CAFs). Here, an assembled organoid model was generated to investigate the mechanism by which the TME regulates angiogenesis in oral squamous cell carcinoma (OSCC). Secretion of vascular endothelial growth factor-A (VEGFA) was analysed to compare the proangiogenic properties of OSCC cells and corresponding CAFs. Cell aggregates consisting of endothelial cells (ECs), CAFs and cancer cells were generated to construct assembled organoids. Nicotinamide N-methyltransferase (NNMT) was pharmacologically or genetically inhibited to block the activation of CAFs. ATAC-seq was employed to test the transcriptional network of fibroblasts overexpressing NNMT. Compared with cancer cells, CAFs secreted more VEGFA. Coculture with CAFs more effectively promoted the sprouting of ECs. Blockade of CAF activation via inhibition of NNMT drastically reduced the expression of CD31 in the assembled organoids. Overexpression of NNMT enhanced the transcription of genes related to angiogenesis in fibroblasts. Specifically, NNMT orchestrated the enrichment of the transcription factor JUNB at the promoter of VEGFA. We clarify that stromal NNMT enables the steady reproduction of angiogenesis in assembled oral cancer organoids, providing a novel target for exploiting antiangiogenic therapy.

Abstract 67: Predicting colorectal patient prognoses by functional characterisation of heterogeneous cell types and their spatial interaction using a new technique: Whole slide imaging mass cytometry

Abstract Immunotherapies stimulate T cell function in the tumor microenvironment (TME). CD3+ and CD8+ T cell infiltration can predict disease recurrence in colorectal cancer (CRC) patients and has been validated as Immunoscore® (IS). The TME comprises multiple heterogeneous populations of immune cells, including T cells and cancer-associated fibroblasts (CAFs); CRC tumors are also morphologically diverse. Standard BioTools™ (SBI) has developed a novel whole slide Imaging Mass Cytometry™ technique that allows complete analysis of cell population heterogeneity in the TME using &amp;gt;40 markers. We hypothesised there are multiple heterogeneous subtypes of CAFs within CRC tumors and that they modulate T cell infiltration and patient outcome. In collaboration with SBI, we studied a cohort of CRC patients stratified based on T cell infiltrate and disease recurrence. We identified multiple CAF populations that interact with tumor-infiltrating T cell populations. Slides from 10 CRC patients with high or low CD3+CD8+ T cell infiltrate (IS) were stained with a panel encompassing markers for CAF and T cell heterogeneity, function, and metabolism. Slides were imaged using the Hyperion XTi™ Imaging System at SBI. Using a scan mode called Preview Mode, the whole slide was imaged in minutes to identify CAF and T cell phenotypic regions of interest. Next, the same slide was imaged at 1 μm resolution using Cell Mode (CM). Finally, for each sample, an additional serial section was imaged using a slightly lower resolution called Tissue Mode (TM). TM allowed us to image the samples in several hours instead of days while capturing the full heterogeneity of the entire tissue sample. We used clustering to identify phenotype clusters and neighbourhood analysis to study spatial enrichment. We also compared results between CM and TM. We report: Identification of heterogeneous CAF subtypes in CRC patients with high or low IS; determination of CAF populations that spatially interact with T cells in patients with high or low IS; and examination of consistent spatial interactions between CAF and T cells in CRC, and whether these interactions differ in patients with high or low IS. TM revealed several heterogeneous cellular populations that differed between patients with high or low IS; importantly, low IS tumors had more podoplanin+αSMA+ CAF populations and fewer podoplanin+αSMA- cell populations compared with high IS tumors. High IS tumors had more PD-1+CD8+ T cells compared with low IS tumors; these PD-1+CD8+ T cells spatially interacted with podoplanin+αSMA- CAF populations in high IS tumors. CAF-PD-1+CD8+ T cell interactions were absent from low IS tumors. Thus, CAF-PD-1+CD8+ T cell interactions are important in CRC patients with high IS, and these cellular interactions require further investigation. Citation Format: Rory M. Costello, Sonya Fenton, Helen McGuire, Liang Lim, David Howell, Qanber Raza, Jyh Yun Chwee, Roslyn Kemp. Predicting colorectal patient prognoses by functional characterisation of heterogeneous cell types and their spatial interaction using a new technique: Whole slide imaging mass cytometry [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 67.

Abstract 1585: Tenascin C in pancreatic cancer-associated fibroblasts enhances epithelial mesenchymal transition and is associated with resistance to immune checkpoint inhibitor

Abstract Backgrounds: Tenascin C (TNC) is an extracellular matrix glycoprotein that is abundantly expressed in cancer stroma and its overexpression correlates with tumor progression in various types of cancer including pancreatic adenocarcinoma (PAAD). However, the role of TNC in regards to response to immune checkpoint inhibitor (ICI) still remains to be identified. In this study, we aimed to investigate the potential involvement of TNC in the response to ICI among PAAD patients. Materials and Methods: Transcriptomic profiles were downloaded by TCGA and GTEx database and TNC mRNA levels were compared between tumors and normal tissues. These transcriptomic data were deconvoluted using CIBERSORTx to identify specific enriched pathways in cancer cells and cancer-associated fibroblasts (CAFs) in PAAD patients. Bioinformatic programs were used to predict paracrine communications between cancer cells and cancer-associated fibroblasts (CAFs), and the Tumor Immune Dysfunction and Exclusion (TIDE) score was calculated to predict response to ICI treatment in PAAD patients. An independent immunotherapeutic cohort was applied to validate prediction values for immunotherapy. Results: TNC mRNA levels were significantly upregulated in tumors compared to normal tissues in eight cancer types including PAAD. TNC-high (TNC-H) PAAD patients (n = 138) had significant shorter overall survival than TNC-low (TNC-L) patients (n = 40) (p = 0.0125, Log-rank test). Using CIBERSORTx, TNC was estimated to be predominantly expressed in CAFs in PAAD. In pathway analysis, both cancer cells and CAFs had significant enrichments of the epithelial mesenchymal transition pathway in TNC-H patients. Ligand-receptor interaction analysis showed that TNC produced in CAFs had the highest interaction potentials to bind to integrin families such as ITGαV and ITGβ3 in cancer cells. In an independent immunotherapy data cohort, patients with TNC-H and ITGαV-high or ITGβ3-high expression were associated with poor response to ICI treatment and had shorter overall survival than those with both low expressions. Conclusions: In conclusion, these findings suggest that TNC-high CAFs can play a crucial role in tumor progression and resistance to ICI therapy in PAAD patients, and targeting TNC and its interactions with cancer cells may provide a potential strategy for improving the efficacy of ICI therapy in PAAD. Citation Format: Satoru Furuhashi, Yoshifumi Morita, Akio Matsumoto, Shinya Ida, Ryuta Muraki, Ryo Kitajima, Makoto Takeda, Hirotoshi Kikuchi, Yoshihiro Hiramatsu, Hiroya Takeuchi. Tenascin C in pancreatic cancer-associated fibroblasts enhances epithelial mesenchymal transition and is associated with resistance to immune checkpoint inhibitor [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 1585.

Abstract 3155: Metabolic stress induces an intrinsic inflammatory CAF phenotype that regulates stromal architecture in pancreatic cancer

Abstract Macropinocytosis is a critical amino acid supply pathway that fuels the metabolism of tumor cells and cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC). PDAC tumors are deficient in glutamine, a critical amino acid that tumor cells and CAFs use to sustain their cellular fitness. We have previously shown that both PDAC cells and CAFs stimulate macropinocytosis as an adaptive response to glutamine depletion. How these linked metabolic mechanisms might co-operate to control stromal architecture remains elusive. In this study, we employed glutamine starvation and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a selective inhibitor of macropinocytosis, to investigate how metabolic stress impacts the tumor microenvironment. We find that concomitant suppression of glutamine metabolism and macropinocytosis synergizes to drive an intrinsic inflammatory CAF phenotype that requires metabolic stress-induced ERK/MAPK signaling. Utilizing immunocompetent syngeneic mouse models of PDAC, we find that blocking macropinocytosis in these glutamine-depleted tumors diminishes tumor growth and reorganizes the tumor stroma by decreasing collagen deposition, increasing vasculature expansion and enhancing the infiltration of both CD8+ and CD4+ T cells. Alterations in the stromal landscape under these conditions are linked to the enhancement of CAFs that display an inflammatory signature while simultaneously suppressing more myofibroblastic identities within the tumor. Interestingly, we find that these effects on the tumor microenvironment can be harnessed to improve therapeutic responses. We determine that combining macropinocytosis inhibition with immune checkpoint blockade reduces PDAC tumor burden and significantly inhibits metastasis. We also find that macropinocytosis inhibition enhances drug delivery, thereby improving the efficacy of gemcitabine chemotherapy. Our findings demonstrate that intratumoral metabolic stress plays a critical part in preserving and modulating the tumor stroma. Citation Format: Yijuan Zhang, Swetha Maganti, Jennifer Hope, Li Lin, Cheska Marie Galapate, Florent Carrette, Linda Bradley, Cosimo Commisso. Metabolic stress induces an intrinsic inflammatory CAF phenotype that regulates stromal architecture in pancreatic 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 3155.

Abstract 1155: Spatial colocalization and molecular crosstalk of myofibroblastic CAFs and tumor cells shape lymph node metastasis in oral squamous cell carcinoma

Abstract Lymph node metastasis (LNM) is a critical prognostic factor for patients with oral squamous cell carcinoma (OSCC). While previous research has implicated partial epithelial-to-mesenchymal transition (p-EMT) of tumor cells and cancer-associated fibroblasts (CAFs), specifically myofibroblastic CAFs (myCAFs), in LNM, the underlying molecular mechanisms remain poorly understood. Furthermore, the lack of diagnostic and prognostic biomarkers related to LNM has impeded the improvement in clinical outcomes for patients with OSCC. Here, we conducted a comprehensive molecular analysis integrating original and publicly available OSCC data of the bulk genome and transcriptome analyses, single-cell transcriptome profiling, and spatial transcriptome analyses. We found that myCAFs were both quantitatively and functionally activated in LNM-positive samples and spatially colocalized with OSCC cells within the invasive tumor front (ITF), providing a niche that may facilitate LNM. We validated spatial colocalization of myCAFs and OSCC cells using 50 additional Immunohistochemistry (IHC) samples. Mechanistically, myCAFs provided increased extracellular matrix (ECM) signals, such as COLLAGEN, Fibronectin1(FN1), and LAMININ to OSCC cells. Consequently, OSCC cells upregulate ECM receptors such as αβ integrins (ITGA-B), syndecans (SDC), and CD44, leading to upregulating stemness-related genes in OSCC cells. Further analysis using CRISPRGeneEffect data from DepMap found that ITGB1, ITGA3, and CD44 are among the most essential genes for the survival and growth of OSCC cells. By analyzing the spatial transcriptomic data of matched primary tumor sites (HUH001-P1 and HUH001-P2) and metastatic site (HUH001-met) from the same patient, we identified the origins of metastatic cells in the primary tumor site. We further extracted a 23-gene signature from the metastatic origin in ITF, where OSCC and myCAFs colocalize. This spatially-resolved 23-gene signature predicted LNM status and poor overall survival (OS) in clinical OSCC patients. The prognostic significance of the 23-gene signature we extracted from the spatial analysis was also validated in separate clinical cohorts from TCGA study and two other microarray datasets. Our findings provide novel insight into the molecular crosstalk between myCAFs and OSCC cells that facilitates LNM and identifies clinically valuable biomarkers for better prognostication in patients with OSCC. Citation Format: Ken Furudate, Shuya Kasai, Tadashi Yoshizawa, Yuya Sasaki, Kohei Fujikura, Shintaro Goto, Ryohei Ito, Tomoyuki Tanaka, Hiroshi Kijima, Kosei Kubota, Ken Itoh, Wataru Kobayashi, Koichi Takahashi. Spatial colocalization and molecular crosstalk of myofibroblastic CAFs and tumor cells shape lymph node metastasis in oral squamous cell carcinoma [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 1155.

Abstract C044: Macropinocytic stress reprograms cancer-associated fibroblasts and enhances therapeutic responses in pancreatic cancer

Abstract Macropinocytosis is a fundamental metabolic pathway that has been demonstrated to support the survival and function of tumor cells and cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC) through in vitro studies and xenograft mouse models. However, the impact of macropinocytosis on the PDAC tumor microenvironment in an immune-competent setting remains poorly understood. In this study, we utilized the selective inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) to block macropinocytosis in both tumor cells and CAFs in syngeneic PDAC mouse models. We observed that EIPA treatment inhibited PDAC tumor growth, which was accompanied by reduced tumor proliferation and increased cell apoptosis. Moreover, EIPA treatment decreased the presence of myofibroblastic CAFs and collagen deposition within PDAC tumors, while also promoting the expansion of tumor vasculature. By modifying the tumor stroma, EIPA priming enhanced drug delivery and improved the effectiveness of PDAC chemotherapy. Interestingly, immune profiling of PDAC tumors revealed that EIPA administration specifically increased the infiltration of CD8+ and CD4+ T cells. We discovered that EIPA induced changes in the cytokine/chemokine landscape within tumors by triggering an inflammatory CAF (iCAF)-like phenotype. Mechanistically, we discerned that inhibition of macropinocytosis triggers metabolic stress that in turn activates stress-responsive transcription factors and promotes the expression of early-phase proinflammatory cytokines and chemokines in mouse CAFs. Notably, combination treatment with EIPA and immune checkpoint blockade (ICB) reduced PDAC tumor burden and significantly inhibited tumor metastasis. Overall, our findings establish that macropinocytosis functions to support the barrier function of the stroma by maintaining fibroblastic identity and provide further evidence that inhibition of macropinocytosis represents a promising therapeutic modality in PDAC. Citation Format: Yijuan Zhang, Swetha Maganti, Jennifer Hope, Li Ling, Cheska Marie Galapate, Florent Carrette, Linda Bradley, Cosimo Commisso. Macropinocytic stress reprograms cancer-associated fibroblasts and enhances therapeutic responses in pancreatic cancer [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 C044.

Spatial profiling of cancer-associated fibroblasts of sporadic early onset colon cancer microenvironment

The incidence of sporadic early-onset colon cancer (EOCC) has increased worldwide. The molecular mechanisms in the tumor and the tumor microenvironment (TME) in EOCC are not fully understood. The aim of this study is to unravel unique spatial transcriptomic and proteomic profiles in tumor epithelial cells and cancer-associated fibroblasts (CAFs). Here, we divide the sporadic colon cancer tissue samples with transcriptomic data into patients diagnosed with EOCC (<50 yrs) and late-onset colon cancer (LOCC, ≥50 yrs) and then, analyze the data using CIBERSORTx deconvolution software. EOCC tumors are more enriched in CAFs with fibroblast associated protein positive expression (FAP(+)) than LOCC tumors. EOCC patients with higher FAP mRNA levels in CAFs have shorter OS (Log-rank test, p < 0.029). Spatial transcriptomic analysis of 112 areas of interest, using NanoString GeoMx digital spatial profiling, demonstrate that FAP(+) CAFs at the EOCC tumor invasive margin show a significant upregulation of WNT signaling and higher mRNA/protein levels of fibroblast growth factor 20 (FGF20). Tumor epithelial cells at tumor invasive margin of EOCC tumors neighboring FAP(+) CAFs show significantly higher mRNA/protein levels of fibroblast growth factor receptor (FGFR2) and PI3K/Akt signaling activation. NichNET analysis show a potential interaction between FGF20 and FGFFR2. The role of FGF20 in activating FGFR2/pFGFR2 and AKT/pAKT was validated in-vitro. In conclusion, we identify a unique FAP(+) CAF population that showed WNT signaling upregulation and increased FGF20 levels; while neighbor tumor cells show the upregulation/activation of FGFR2-PI3K/Akt signaling at the tumor invasive margin of EOCC tumors.

Musashi-2 in cancer-associated fibroblasts promotes non-small cell lung cancer metastasis through paracrine IL-6-driven epithelial-mesenchymal transition

BackgroundLung cancer, the most common cause of cancer-related mortality worldwide, is predominantly associated with advanced/metastatic disease. The interaction between tumor cells and cancer-associated fibroblasts (CAFs) in tumor microenvironment is known to be essential for regulating tumor progression and metastasis, but the underlying mechanisms, particularly the role of RNA-binding protein Musashi-2 (MSI2) in CAFs in promoting non-small cell lung cancer (NSCLC) invasiveness and metastatic spread, remain obscure.MethodsGenomic and proteomic database analyses were performed to evaluate the potential clinical significance of MSI2 in NSCLC tumor and stromal clinical specimens. Molecular approaches were used to modify MSI2 in CAFs and determine its functional role in NSCLC cell motility in vitro using 2D and 3D models, and in metastasis in a xenograft mouse model using live-cell imaging.ResultsMSI2, both gene and protein, is upregulated in NSCLC tissues and is associated with poor prognosis and high metastatic risk in patients. Interestingly, MSI2 is also upregulated in NSCLC stroma and activated fibroblasts, including CAFs. Depletion of MSI2 in CAFs by CRISPR-Cas9 strongly inhibits NSCLC cell migration and invasion in vitro, and attenuates local and distant metastatic spread of NSCLC cells in vivo. The crosstalk between CAFs and NSCLC cells occurs via paracrine signaling, which is regulated by MSI2 in CAFs via IL-6. The secreted IL-6 promotes epithelial-mesenchymal transition in NSCLC cells, which drives metastasis.ConclusionOur findings reveal for the first time that MSI2 in CAFs is important in CAF-mediated NSCLC cell invasiveness and metastasis via IL-6 paracrine signaling. Therefore, targeting the MSI2/IL-6 axis in CAFs could be effective in combating NSCLC metastasis.

Optimization of a Three-Dimensional Culturing Method for Assessing the Impact of Cisplatin on Notch Signaling in Head and Neck Squamous Cell Carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, with cisplatin being a primary treatment approach. However, drug resistance and therapy failure pose a significant challenge, affecting nearly 50% of patients over time. This research had two aims: (1) to optimize a 3D cell-culture method for assessing the interplay between tumor cells and cancer-associated fibroblasts (CAFs) in vitro; and (2) to study how cisplatin impacts the Notch pathway, particularly considering the role of CAFs. Using our optimized "3D sheet model" approach, we tested two HNSCC cell lines with different cisplatin sensitivities and moderate, non-mutated NOTCH1 and -3 expressions. Combining cisplatin with a γ-secretase inhibitor (crenigacestat) increased sensitivity and induced cell death in the less sensitive cell line, while cisplatin alone was more effective in the moderately sensitive line and sensitivity decreased with the Notch inhibitor. Cisplatin boosted the expression of core Notch signaling proteins in 3D monocultures of both lines, which was counteracted by crenigacestat. In contrast, the presence of patient-derived CAFs mitigated effects and protected both cell lines from cisplatin toxicity. Elevated NOTCH1 and NOTCH3 protein levels were consistently correlated with reduced cisplatin sensitivity and increased cell survival. Additionally, the Notch ligand JAG2 had additional, protective effects reducing cell death from cisplatin exposure. In summary, we observed an inverse relationship between NOTCH1 and NOTCH3 levels and cisplatin responsiveness, overall protective effects by CAFs, and a potential link between JAG2 expression with tumor cell survival.

Integrating single-cell and spatial transcriptomics to elucidate the crosstalk between cancer-associated fibroblasts and cancer cells in hepatocellular carcinoma with spleen-deficiency syndrome

Background and aimMost patients with hepatocellular carcinoma (HCC) in China have been diagnosed with spleen deficiency syndrome (SDS), which accelerates the progression of HCC by disrupting the tumor microenvironment homeostasis. This study aimed to investigate the intercellular crosstalk in HCC with SDS. Experimental procedureAn HCC-SDS mouse model was established using orthotopic HCC transplantation based on reserpine-induced SDS. Single-cell data analysis and cancer cell prediction were conducted using Seurat and CopyKAT package, respectively. Intercellular interactions were explored using CellPhoneDB and CellChat and subsequently validated using co-culture assays, ELISA and histological staining. We performed pathway activity analysis using gene set variation analysis and the Seurat package. The extracellular matrix (ECM) remodeling was assessed using a gel contraction assay, atomic force microscopy, and Sirius red staining. The deconvolution of the spatial transcriptomics data using the “CARD” package based on single-cell data. Results and conclusionWe successfully established the HCC-SDS mouse model. Twenty-nine clusters were identified. The interactions between cancer cells and cancer-associated fibroblasts (CAFs) were significantly enhanced via platelet-derived growth factor (PDGF) signaling in HCC-SDS. CAFs recruited in HCC-SDS lead to ECM remodeling and the activation of TGF-β signaling pathway. Deconvolution of the spatial transcriptome data revealed that CAFs physically surround cancer cells in HCC-SDS. This study reveals that the crosstalk of CAFs-cancer cells is crucial for the tumor-promoting effect of SDS. CAFs recruited by HCC via PDGFA may lead to ECM remodeling through activation of the TGF-β pathway, thereby forming a physical barrier to block immune cell infiltration under SDS.

High-dimensional deconstruction of pancreatic cancer identifies tumor microenvironmental and developmental stemness features that predict survival

Numerous cell states are known to comprise the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the developmental stemness and co-occurrence of these cell states remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) samples (n = 25). We then combined these samples with surgical resection (n = 6) and publicly available samples to increase statistical power (n = 80). Following annotation into 25 distinct cell states, cells were scored for developmental stemness, and a customized version of the Ecotyper tool was used to identify communities of co-occurring cell states in bulk RNA-seq samples (n = 268). We discovered a tumor microenvironmental community comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages, and myofibroblastic cancer-associated fibroblasts associated with especially poor prognosis. We also found a developmental stemness continuum with implications for survival that is present in both malignant cells and cancer-associated fibroblasts (CAFs). We further demonstrated that high-dimensional analyses predictive of survival are feasible using standard-of-care, time-of-diagnosis EUS-FNB specimens. In summary, we identified tumor microenvironmental and developmental stemness characteristics from a high-dimensional gene expression analysis of PDAC using human tissue specimens, including time-of-diagnosis EUS-FNB samples. These reveal new connections between tumor microenvironmental composition, CAF and malignant cell stemness, and patient survival that could lead to better upfront risk stratification and more personalized upfront clinical decision-making.

Spatial transcriptomics analysis of neoadjuvant cabozantinib and nivolumab in advanced hepatocellular carcinoma identifies independent mechanisms of resistance and recurrence

BackgroundNovel immunotherapy combination therapies have improved outcomes for patients with hepatocellular carcinoma (HCC), but responses are limited to a subset of patients. Little is known about the inter- and intra-tumor heterogeneity in cellular signaling networks within the HCC tumor microenvironment (TME) that underlie responses to modern systemic therapy.MethodsWe applied spatial transcriptomics (ST) profiling to characterize the tumor microenvironment in HCC resection specimens from a prospective clinical trial of neoadjuvant cabozantinib, a multi-tyrosine kinase inhibitor that primarily blocks VEGF, and nivolumab, a PD-1 inhibitor in which 5 out of 15 patients were found to have a pathologic response at the time of resection.ResultsST profiling demonstrated that the TME of responding tumors was enriched for immune cells and cancer-associated fibroblasts (CAF) with pro-inflammatory signaling relative to the non-responders. The enriched cancer-immune interactions in responding tumors are characterized by activation of the PAX5 module, a known regulator of B cell maturation, which colocalized with spots with increased B cell marker expression suggesting strong activity of these cells. HCC-CAF interactions were also enriched in the responding tumors and were associated with extracellular matrix (ECM) remodeling as there was high activation of FOS and JUN in CAFs adjacent to the tumor. The ECM remodeling is consistent with proliferative fibrosis in association with immune-mediated tumor regression. Among the patients with major pathologic responses, a single patient experienced early HCC recurrence. ST analysis of this clinical outlier demonstrated marked tumor heterogeneity, with a distinctive immune-poor tumor region that resembles the non-responding TME across patients and was characterized by HCC-CAF interactions and expression of cancer stem cell markers, potentially mediating early tumor immune escape and recurrence in this patient.ConclusionsThese data show that responses to modern systemic therapy in HCC are associated with distinctive molecular and cellular landscapes and provide new targets to enhance and prolong responses to systemic therapy in HCC.

Amphiregulin couples IL1RL1+ regulatory T cells and cancer-associated fibroblasts to impede antitumor immunity.

Regulatory T (Treg) cells and cancer-associated fibroblasts (CAFs) jointly promote tumor immune tolerance and tumorigenesis. The molecular apparatus that drives Treg cell and CAF coordination in the tumor microenvironment (TME) remains elusive. Interleukin 33 (IL-33) has been shown to enhance fibrosis and IL1RL1+ Treg cell accumulation during tumorigenesis and tissue repair. We demonstrated that IL1RL1 signaling in Treg cells greatly dampened the antitumor activity of both IL-33 and PD-1 blockade. Whole tumor single-cell RNA sequencing (scRNA-seq) analysis and blockade experiments revealed that the amphiregulin (AREG)-epidermal growth factor receptor (EGFR) axis mediated cross-talk between IL1RL1+ Treg cells and CAFs. We further demonstrated that the AREG/EGFR axis enables Treg cells to promote a profibrotic and immunosuppressive functional state of CAFs. Moreover, AREG mAbs and IL-33 concertedly inhibited tumor growth. Our study reveals a previously unidentified AREG/EGFR-mediated Treg/CAF coupling that controls the bifurcation of fibroblast functional states and is a critical barrier for cancer immunotherapy.

Cancer-Associated Fibroblasts Influence Survival in Pleural Mesothelioma: Digital Gene Expression Analysis and Supervised Machine Learning Model.

The exact mechanism of desmoplastic stromal reaction (DSR) formation is still unclear. The interaction between cancer cells and cancer-associated fibroblasts (CAFs) has an important role in tumor progression, while stromal changes are a poor prognostic factor in pleural mesothelioma (PM). We aimed to assess the impact of CAFs paracrine signaling within the tumor microenvironment and the DSR presence on survival, in a cohort of 77 PM patients. DSR formation was evaluated morphologically and by immunohistochemistry for Fibroblast activation protein alpha (FAP). Digital gene expression was analyzed using a custom-designed CodeSet (NanoString). Decision-tree-based analysis using the "conditional inference tree" (CIT) machine learning algorithm was performed on the obtained results. A significant association between FAP gene expression levels and the appearance of DSR was found (p = 0.025). DSR-high samples demonstrated a statistically significant prolonged median survival time. The elevated expression of MYT1, KDR, PIK3R1, PIK3R4, and SOS1 was associated with shortened OS, whereas the upregulation of VEGFC, FAP, and CDK4 was associated with prolonged OS. CIT revealed a three-tier system based on FAP, NF1, and RPTOR expressions. We could outline the prognostic value of CAFs-induced PI3K signaling pathway activation together with FAP-dependent CDK4 mediated cell cycle progression in PM, where prognostic and predictive biomarkers are urgently needed to introduce new therapeutic strategies.

Inkjet-printed morphogenesis of tumor-stroma interface using bi-cellular bioinks of collagen-poly(N-isopropyl acrylamide-co-methyl methacrylate) mixture

Recent advances in biomaterials and 3D printing/culture methods enable various tissue-engineered tumor models. However, it is still challenging to achieve native tumor-like characteristics due to lower cell density than native tissues and prolonged culture duration for maturation. Here, we report a new method to create tumoroids with a mechanically active tumor-stroma interface at extremely high cell density. This method, named “inkjet-printed morphogenesis” (iPM) of the tumor-stroma interface, is based on a hypothesis that cellular contractile force can significantly remodel the cell-laden polymer matrix to form densely-packed tissue-like constructs. Thus, differential cell-derived compaction of tumor cells and cancer-associated fibroblasts (CAFs) can be used to build a mechanically active tumor-stroma interface. In this methods, two kinds of bioinks are prepared, in which tumor cells and CAFs are suspended respectively in the mixture of collagen and poly (N-isopropyl acrylamide-co-methyl methacrylate) solution. These two cellular inks are inkjet-printed in multi-line or multi-layer patterns. As a result of cell-derived compaction, the resulting structure forms tumoroids with mechanically active tumor-stroma interface at extremely high cell density. We further test our working hypothesis that the morphogenesis can be controlled by manipulating the force balance between cellular contractile force and matrix stiffness. Furthermore, this new concept of “morphogenetic printing” is demonstrated to create more complex structures beyond current 3D bioprinting techniques.

Schwann cells regulate tumor cells and cancer-associated fibroblasts in the pancreatic ductal adenocarcinoma microenvironment

Neuropathy is a feature more frequently observed in pancreatic ductal adenocarcinoma (PDAC) than other tumors. Schwann cells, the most prevalent cell type in peripheral nerves, migrate toward tumor cells and associate with poor prognosis in PDAC. To unveil the effects of Schwann cells on the neuro-stroma niche, here we perform single-cell RNA-sequencing and microarray-based spatial transcriptome analysis of PDAC tissues. Results suggest that Schwann cells may drive tumor cells and cancer-associated fibroblasts (CAFs) to more malignant subtypes: basal-like and inflammatory CAFs (iCAFs), respectively. Moreover, in vitro and in vivo assays demonstrate that Schwann cells enhance the proliferation and migration of PDAC cells via Midkine signaling and promote the switch of CAFs to iCAFs via interleukin-1α. Culture of tumor cells and CAFs with Schwann cells conditioned medium accelerates PDAC progression. Thus, we reveal that Schwann cells induce malignant subtypes of tumor cells and CAFs in the PDAC milieu.

The Ephrin tyrosine kinase a3 (EphA3) is a novel mediator of RAGE-prompted motility of breast cancer cells

BackgroundThe receptor for advanced glycation-end products (RAGE) and its ligands have been implicated in obesity and associated inflammatory processes as well as in metabolic alterations like diabetes. In addition, RAGE-mediated signaling has been reported to contribute to the metastatic progression of breast cancer (BC), although mechanistic insights are still required. Here, we provide novel findings regarding the transcriptomic landscape and the molecular events through which RAGE may prompt aggressive features in estrogen receptor (ER)-positive BC.MethodsMCF7 and T47D BC cells stably overexpressing human RAGE were used as a model system to evaluate important changes like cell protrusions, migration, invasion and colony formation both in vitro through scanning electron microscopy, clonogenic, migration and invasion assays and in vivo through zebrafish xenografts experiments. The whole transcriptome of RAGE-overexpressing BC cells was screened by high-throughput RNA sequencing. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses allowed the prediction of potential functions of differentially expressed genes (DEGs). Flow cytometry, real time-PCR, chromatin immunoprecipitation, immunofluorescence and western blot assays were performed to investigate the molecular network involved in the regulation of a novel RAGE target gene namely EphA3. The clinical significance of EphA3 was explored in the TCGA cohort of patients through the survivALL package, whereas the pro-migratory role of EphA3 signaling was ascertained in both BC cells and cancer-associated fibroblasts (CAFs). Statistical analysis was performed by t-tests.ResultsRNA-seq findings and GSEA analysis revealed that RAGE overexpression leads to a motility-related gene signature in ER-positive BC cells. Accordingly, we found that RAGE-overexpressing BC cells exhibit long filopodia-like membrane protrusions as well as an enhanced dissemination potential, as determined by the diverse experimental assays. Mechanistically, we established for the first time that EphA3 signaling may act as a physical mediator of BC cells and CAFs motility through both homotypic and heterotypic interactions.ConclusionsOur data demonstrate that RAGE up-regulation leads to migratory ability in ER-positive BC cells. Noteworthy, our findings suggest that EphA3 may be considered as a novel RAGE target gene facilitating BC invasion and scattering from the primary tumor mass. Overall, the current results may provide useful insights for more comprehensive therapeutic approaches in BC, particularly in obese and diabetic patients that are characterized by high RAGE levels.

Expression Profile of Microenvironmental Factors in the Interface Zone of Colorectal Cancer: Histological-Stromal Biomarkers and Cancer Cell-Cancer-Associated Fibroblast-Related Proteins Combined for the Assessment of Tumor Progression

Introduction: The characterization of tumor microenvironment (TME) related factors and their impact on tumor progression have attracted much interest. We investigated cancer cells and cancer-associated fibroblasts (CAFs) to evaluate biomarkers that are associated with neoplastic progression, observing them in different interface zones of colorectal cancer. Methods: On 357 CRC tissue microarrays, using immunohistochemistry, we examined the associations of podoplanin and α-SMA expressed in cancer cells and CAFs and evaluated them in different areas: tumor core, invasive front, tumor budding, tumor-stroma ratio (TSR) scoring, and desmoplastic stroma. Results: CAFs expressing α-SMA were found in more than 90% of the cases. Podoplanin+ was detected in cancer cells and CAFs, with positivities of 38.6% and 70%, respectively. Higher α-SMA+ CAFs and podoplanin+ cancer cells were observed predominantly at the TSR score area: 94.3% and 64.3% of cases, respectively. The status of podoplanin in CAFs+ was higher in the desmoplastic area (71.6%). Stroma-high tumors showed increased expression of α-SMA and podoplanin in comparison with stroma-low tumors. The status of podoplanin in cancer cells was observed in association with lymphatic invasion and distant metastasis. Conclusion: The substance of the CRC was composed predominantly of the surrounding stroma-α-SMA+ CAFs. Podoplanin expressed in the prognosticator zones was associated with unfavorable pathological features. The combination of histologic and protein-related biomarkers can result in a tool for the stratification of patients with CRC.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.