Fibrotic Tumor Research Articles

Abstract 737: Arrays of 3D ECM-embedded fibroblast clusters for characterization of ECM remodeling by primary cancer associated fibroblasts and for evaluation of drugs attenuating fibrotic ECM remodeling

Abstract Chronic fibrosis can lead to organ failure and is also associated with cancer, where the tumor is interpreted as a chronic wound, and the fibrotic tumor environment drives disease progression. Fibrosis occurs in an inflammatory environment where macrophages are activated, and fibroblasts transdifferentiate into myofibroblasts. Activated macrophages stimulate the myofibroblasts to produce excessive amounts of collagen-rich ECM. Changes in proteolytic activity and high contractile forces that myofibroblasts apply onto their tissue environment, further lead to extensive remodeling of the healthy tissue matrix into a scar matrix. Similar activity is displayed by cancer-associated fibroblasts (CAFs) in tumors. Intervening with the excessive ECM remodeling by activated fibroblasts represents a candidate therapeutic strategy to normalize the architecture of fibrotic and malignant tissues. We developed a screening platform using an assay based on automated image guided injection of clusters of fibroblasts in wells of multi-well plates preloaded with a collagen-rich ECM network. The identical x-y-z position, spacing, and size of the ECM-embedded fibroblast clusters in each well facilitates automated real time confocal microscopy and quantitative image analysis algorithms. We use this setup for automated quantitative analysis of ECM remodeling activity of CAFs obtained from early-stage colorectal cancer patients. We find that CAFs from these early-stage lesions display increased ECM remodeling capacity as compared to patient matched normal fibroblasts. We then use the same setup to screen a series of compounds for their ability to attenuate ECM remodeling triggered by TGFb-activated primary human fibroblasts. We detect known and novel pathways, and we identify small molecule inhibitors with a promising efficacy versus toxicity profile. Lastly, we detect a previously unknown concentration-dependent side effect of ALK5 inhibitors that leads to the identification of a novel profibrotic pathway and indicates that caution is warranted for the clinical application of ALK5 inhibitors. Citation Format: Erik H. J. Danen. Arrays of 3D ECM-embedded fibroblast clusters for characterization of ECM remodeling by primary cancer associated fibroblasts and for evaluation of drugs attenuating fibrotic ECM remodeling [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 737.

Breaking Physical Barrier of Fibrotic Breast Cancer for Photodynamic Immunotherapy by Remodeling Tumor Extracellular Matrix and Reprogramming Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) assist in breast cancer (BRCA) invasion and immune resistance by overproduction of extracellular matrix (ECM). Herein, we develop FPC@S, a photodynamic immunomodulator that targets the ECM, to improve the photodynamic immunotherapy for fibrotic BRCA. FPC@S combines a tumor ECM-targeting peptide, a photosensitizer (protoporphyrin IX) and an antifibrotic drug (SIS3). After anchoring to the ECM, FPC@S causes ECM remodeling and BRCA cell death by generating reactive oxygen species (ROS) in situ. Interestingly, the ROS-mediated ECM remodeling can normalize the tumor blood vessel to improve hypoxia and in turn facilitate more ROS production. Besides, upon the acidic tumor microenvironment, FPC@S will release SIS3 for reprograming CAFs to reduce their activity but not kill them, thus inhibiting fibrosis while preventing BRCA metastasis. The natural physical barrier formed by the dense ECM is consequently eliminated in fibrotic BRCA, allowing the drugs and immune cells to penetrate deep into tumors and have better efficacy. Furthermore, FPC@S can stimulate the immune system and effectively suppress primary, distant and metastatic tumors by combining with immune checkpoint blockade therapy. This study provides different insights for the development of fibrotic tumor targeted delivery systems and exploration of synergistic immunotherapeutic mechanisms against aggressive BRCA.

Aberrant TIMP-1 production in tumor-associated fibroblasts drives the selective benefits of nintedanib in lung adenocarcinoma.

The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with invitro and invivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors.

Mechanoregulation and function of calponin and transgelin.

It is well known that chemical energy can be converted to mechanical force in biological systems by motor proteins such as myosin ATPase. It is also broadly observed that constant/static mechanical signals potently induce cellular responses. However, the mechanisms that cells sense and convert the mechanical force into biochemical signals are not well understood. Calponin and transgelin are a family of homologous proteins that participate in the regulation of actin-activated myosin motor activity. An isoform of calponin, calponin 2, has been shown to regulate cytoskeleton-based cell motility functions under mechanical signaling. The expression of the calponin 2 gene and the turnover of calponin 2 protein are both under mechanoregulation. The regulation and function of calponin 2 has physiological and pathological significance, as shown in platelet adhesion, inflammatory arthritis, arterial atherosclerosis, calcific aortic valve disease, post-surgical fibrotic peritoneal adhesion, chronic proteinuria, ovarian insufficiency, and tumor metastasis. The levels of calponin 2 vary in different cell types, reflecting adaptations to specific tissue environments and functional states. The present review focuses on the mechanoregulation of calponin and transgelin family proteins to explore how cells sense steady tension and convert the force signal to biochemical activities. Our objective is to present a current knowledge basis for further investigations to establish the function and mechanisms of calponin and transgelin in cellular mechanoregulation.

Disruption of Super-Enhancers in Activated Pancreatic Stellate Cells Facilitates Chemotherapy and Immunotherapy in Pancreatic Cancer.

One major obstacle in the drug treatment of pancreatic ductal adenocarcinoma (PDAC) is its highly fibrotic tumor microenvironment, which is replete with activated pancreatic stellate cells (a-PSCs). These a-PSCs generate abundant extracellular matrix and secrete various cytokines to form biophysical and biochemical barriers, impeding drug access to tumor tissues. Therefore, it is imperative to develop a strategy for reversing PSC activation and thereby removing the barriers to facilitate PDAC drug treatment. Herein, by integrating chromatin immunoprecipitation (ChIP)-seq, Assays for Transposase-Accessible Chromatin (ATAC)-seq, and RNA-seq techniques, this work reveals that super-enhancers (SEs) promote the expression of various genes involved in PSC activation. Disruption of SE-associated transcription with JQ1 reverses the activated phenotype of a-PSCs and decreases stromal fibrosis in both orthotopic and patient-derived xenograft (PDX) models. More importantly, disruption of SEs by JQ1 treatments promotes vascularization, facilitates drug delivery, and alters the immune landscape in PDAC, thereby improving the efficacies of both chemotherapy (with gemcitabine) and immunotherapy (with IL-12). In summary, this study not only elucidates the contribution of SEs of a-PSCs in shaping the PDAC tumor microenvironment but also highlights that targeting SEs in a-PSCs may become a gate-opening strategy that benefits PDAC drug therapy by removing stromal barriers.

Magnetic-optical dual-modality imaging monitoring chemotherapy efficacy of pancreatic ductal adenocarcinoma with a low-dose fibronectin-targeting Gd-based contrast agent.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal hypovascular tumor surrounded by dense fibrosis. Albumin-bound paclitaxel and gemcitabine (AG) chemotherapy is the mainstay of PDAC treatment through depleting peritumoral fibrosis and killing tumor cells; however, it remains challenging due to the lack of a noninvasive imaging method evaluating fibrotic changes during AG chemotherapy. In this study, we developed a dual-modality imaging platform that enables noninvasive, dynamic, and quantitative assessment of chemotherapy-induced fibrotic changes through near-infrared fluorescence molecular imaging (FMI) and magnetic resonance imaging (MRI) using an extradomain B fibronectin (EDB-FN)-targeted imaging probe (ZD2-Gd-DOTA-Cy7). The ZD2-Gd-DOTA-Cy7 probe was constructed by conjugating a peptide (Cys-TVRTSAD) to Gd-DOTA and the near-infrared dye Cy7. PDAC murine xenograft models were intravenously injected with ZD2-Gd-DOTA-Cy7 at a Gd concentration of 0.05 mmol/kg or free Cy7 and Gd-DOTA as control. The normalized tumor background ratio (TBR) on FMI and the T1 reduction ratio on MRI were quantitatively analyzed. For models receiving AG chemotherapy or saline, MRI/FMI was performed before and after treatment. Histological analyses were performed for validation. The ZD2-Gd-DOTA-Cy7 concentration showed a linear correlation with the fluorescence intensity and T1 relaxation time in vitro. The optimal imaging time was 30min after injection of the ZD2-Gd-DOTA-Cy7 (0.05 mmol/kg), only half of the clinic dosage of gadolinium. Additionally, ZD2-Gd-DOTA-Cy7 generated a 1.44-fold and 1.90-fold robust contrast enhancement compared with Cy7 (P < 0.05) and Gd-DOTA (P < 0.05), respectively. For AG chemotherapy monitoring, the T1 reduction ratio and normalized TBR in the fibrotic tumor areas were significantly increased by 1.99-fold (P < 0.05) and 1.78-fold (P < 0.05), respectively, in the control group compared with those in the AG group. MRI/FMI with a low dose of ZD2-Gd-DOTA-Cy7 enables sensitive imaging of PDAC and the quantitative assessment of fibrotic changes during AG chemotherapy, which shows potential clinical applications for precise diagnosis, post-treatment monitoring, and disease management.

Abstract A033: Blocking TNF-a producing macrophage activates antitumor immunity in pancreatic cancer via IL33

Abstract Pancreatic ductal adenocarcinoma (PDA) remains resistant to immune therapies, largely due to robustly fibrotic and immunosuppressive tumor microenvironments. It has been postulated that excessive accumulation of immunosuppressive myeloid cells influences immunotherapy resistance and recent studies targeting macrophages in combination with checkpoint blockade have demonstrated promising preclinical results. Yet, our understanding of tumor-associated macrophage (TAM) function, complexity, and diversity in PDA remains limited. Here, analysis of human PDA single cell sequencing data revealed significant macrophage heterogeneity, with bone marrow-derived monocytes serving as the primary source for immunosuppressive TAMs. These bone marrow derived macrophages also served as a primary source of TNF-a. Deletion of Ccr2 in genetically engineered KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mice decreased monocyte recruitment resulting in decreased metastasis, and increased survival. Moreover, intervention studies targeting CCR2 with a new orthosteric inhibitor (CCX598) renders PDA susceptible to checkpoint blockade therapy (CTLA-4 and PD-L1) resulting in reduced metastatic burden and increased survival in KPC mice. Additionally, TNF-a suppresses the expression of alarmin IL-33 in cancer cells, thereby hindering dendritic cell and cytotoxic T cell anti-tumor activity. When put together our data indicate that interventions to disrupt infiltration of immunosuppressive macrophages lead to decreased TNF-a, decreased metastasis and increased survival in two different models suggesting its potential role to overcome barriers to effective immunotherapeutics for PDA. Citation Format: Ajay Dixit, Paolo Provenzano, Aaron Sarver, Jon Zettervall, Huocong Huang, Kexin Zheng, Rolf Brekken. Blocking TNF-a producing macrophage activates antitumor immunity in pancreatic cancer via IL33 [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 A033.

Abstract C046: Enhancing T cell infiltration and migration in the pancreatic tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, with a five-year survival rate only increasing from 6% to 12% in the last decade, indicating an urgent need for new therapeutic approaches. In recent years, immunotherapies like chimeric antigen receptor (CAR)-T cell therapy, have demonstrated promising therapeutic results in treating several hematological malignancies. However, the success has been limited in treating solid tumors, especially for PDAC. Factors contributing to this poor response include poor immunogenicity, highly heterogeneous and robust fibrotic and immunosuppressive tumor microenvironments (TMEs). Here, we used state-of-the-art imaging coupled with quantitative analysis to study dynamic T cell migration behavior in organotypic pancreatic tumor slices harvested from genetically engineered KPC mice. Our data showed that T cell speed and overall motility are both significantly lower in stroma-rich region PDA regions than observations in carcinoma cell-rich regions, where collagen fibers are loosely connected. Next, we utilized stroma-targeted approaches to deplete collagen and hyaluronan, the most abundant ECM components in the TME, to remove the biophysical barriers for effective immune therapy. Indeed, preliminary data demonstrated improved T cell infiltration and migration in the tumor slices in the STT-treated groups compared to the control groups. The observation is also confirmed with a therapeutic cell line generated with mesothelin-specific T cell receptors. Furthermore, we utilized genome engineering to generate CD8+ T cells that more effectively migrate in fibrous TMEs. Time-lapse imaging of the engineered T cells shows an increased 3D migration compared to control groups. Taken together, our data suggest that engineered T cell approaches in combination with stromal targeted therapies to diminish stromal ECM content have the potential to improve immunotherapy by allowing effector T cells to sample the tumor volume more effectively. Citation Format: Hongrong Zhang, Guhan Qian, Zhongming Chen, Branden Moriarity, Ingunn Stromnes, David Odde, Paolo Provenzano. Enhancing T cell infiltration and migration in the pancreatic tumor microenvironment [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 C046.

Abstract B004: EUS guided local administration of large surface area microparticle paclitaxel with neoadjuvant chemotherapy in locally advanced pancreatic cancer: A single center experience

Abstract Background: Locally advanced pancreatic cancer (LAPC) is defined as an unresectable tumor due to extensive vascular involvement without evidence of metastatic spread. Neoadjuvant chemotherapy with leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) or gemcitabine-nab-paclitaxel (gem-nab) are the preferred treatments in LAPC. These treatments downstage LAPC to resectable disease in about 10%–35% of patients resulting in an increase in median survival. However, intravenous paclitaxel's hydrophobic, protein-bound nature, short half-life, and intermittent dosing hamper tumor tissue exposure time, resulting in suboptimal intratumoral bioavailability that may facilitate tumor resistance. Limited downstaging to R0 resection is likely due, in part, to the desmoplastic, fibrotic tumor microenvironment in LAPC and poorly vascularized stroma reducing availability of IV drug to tumor cells. To address this, large surface area microparticle paclitaxel (LSAM-PTX) was developed (NanOlogy LLC) for intratumoral (IT) delivery with a mean particle size (DV50) of about 2.5 µm and 3-fold increase in specific surface area over unprocessed drug. The relatively large particle size facilitates tumor retention, and increased surface area allows continuous drug release in tumor over an extended time, along with negligible systemic exposure. Methods: Data were collected prospectively on 6 of 13 (46%) subjects with LAPC who received EUS-guided fine needle injection (EUS-FNI) of LSAM-PTX in addition to neoadjuvant chemotherapy and subsequently underwent surgery at our institution from 2018 to 2019 (NCT03077685). Each subject underwent two EUS-FNI procedures four weeks apart to deliver IT LSAM-PTX suspension (15 mg/mL) in a volume equivalent to 20% of the tumor volume up to 5 mL of drug. Histopathological and immunophenotypic profiling using multiplex immunofluorescence (mIF) was performed on tumor site tissue from pre-LSAM-PTX biopsies and the resected tumor. Results: The mean duration from EUS-FNI to surgery was 6.91 months (range: 3.68-12.22 months). Histopathology was evaluated from the resected specimens to assess treatment response. Five of six subjects (83%) had R0 resections. Of the five R0 resections, one subject had a pathologic complete response, one had 10% and two had 10-20% viable tumor, and one had a partial response with tumor regression. Focal necrosis was also reported in the lone subject with an R1 resection. Immunophenotypic profiling of tissue showed an increase in adaptive and innate immune cell density, an increase in NK cells in the tumor microenvironment, and a decrease in the myeloid/MDSC populations. Conclusions: EUS-FNI of LSAM-PTX, added to neoadjuvant chemotherapy, was safe and resulted in a significant reduction in tumor volume, along with significant tumor necrosis, and favorable changes in tumor immunophenotypic configuration. These preliminary results suggest that adding LSAM-PTX to neoadjuvant chemotherapy could increase the rate of downstaging of LAPC to resectable disease and improve clinical outcomes. Citation Format: Harishankar Gopakumar, Aqsa Khan, Christina M. Zelt, Ashley Rumple, Mariajose Rojas de Leon, Neil R. Sharma. EUS guided local administration of large surface area microparticle paclitaxel with neoadjuvant chemotherapy in locally advanced pancreatic cancer: A single center experience [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 B004.

Abstract A046: nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma

Abstract The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. Packaging of specific cellular constituents, such as prion protein and specific miRNAs involved in cancer progression, into exosomes are dependent on nSMase2. Employing the KPC mouse model of pancreatic ductal adenocarcinoma (PDA), we demonstrate that nSMase2 regulates exosome secretion and in vivo growth of PDA cells. PDA cell exosomes generated through nSMase2 accelerate PDA progression, and PDA cell exosomes generated independently of nSMase2 are anti-tumorigenic during PDA progression. Homozygous loss of Smpd3 (gene which encodes nSMase2) in KPC mouse pancreatic epithelial cells during development extends survival of KPC mice. KPC; Smpd3f/f mice display less PanIN and tumor burden compared to KPC; Smpd3wt/wt controls. In addition, nSMase2 loss in KPC PDA cells promoted a PDA subtype switch from aggressive basal-like to classical. RNA-sequencing of PDA cell lines generated from pancreatic tumors of end-stage KPC; Smpd3wt/wt, KPC; Smpd3f/wt, and KPC; Smpd3f/f mice demonstrated that nSMase2-generated ceramide is a key regulator of intracellular pathways regulating tumor microenvironment dynamics in PDA cells including fibrosis and integrins. We observed a decrease in extracellular matrix collagen abundance and fewer activated stellate cells and fibroblasts in KPC; Smpd3f/f compared to control KPC; Smpd3wt/wt mouse pancreata. Exosomes isolated from KPC; Smpd3wt/wt PDA cell lines significantly stimulate proliferation of primary murine stellate cells in vitro, while exosomes isolated from KPC; Smpd3f/f PDA cell lines do not. Abrogation of Smpd3 expression also affected immune cell infiltration, as demonstrated by a significant increase in iNOS+ F4/80+ double positive proinflammatory macrophages in KPC; Smpd3f/f pancreata when compared to KPC; Smpd3wt/wt pancreata. Lipidomics analysis of epithelial cell lines generated from end-stage pancreatic tumors of KPC; Smpd3f/f and KPC; Smpd3wt/wt mice demonstrated an alteration in hundreds of lipid species including ceramides, triacylglycerides, sphingomyelins, and phosphatidylcholines. Proteomics and small RNA-seq analysis of exosomes secreted from KPC; Smpd3wt/wt and KPC; Smpd3f/f PDA cell lines demonstrated significant alteration of &amp;gt;700 proteins and 12 miRNAs. Our patient data demonstrate that low nSMase2 expression in treatment naïve primary resected pancreatic tumors significantly correlates with fewer CD31+ endothelial cells. In parallel, loss of Smpd3 resulted in a reduction in CD31+ endothelial cells in pancreatic tumors of KPC; Smpd3f/f mice when compared to KPC; Smpd3wt/wt controls. Thus, nSMase2 is a critical mediator of angiogenesis during PDA progression, which is integral to chemotherapeutic response. Collectively, our data show that nSMase2-generated exosomes promote PDA tumorigenesis, specifically proliferation of stellate cells, which may in turn induce a fibrotic tumor microenvironment that affects vasculature development. Citation Format: Audrey M. Hendley, Atsushi Urano, Xianlu L. Peng, Sudipta Ashe, Tuan A. Phu, Martin Ng, Natanya R. Kerper, David I. Berrios, Jonghyun Lee, David Jin, Gun H. Jang, Oghenekevwe M. Gbenedio, Jeroen P. Roose, Jen J. Yeh, Steven Gallinger, Andrew V. Biankin, Vasilis Ntranos, David K. Chang, David W. Dawson, Grace E. Kim, Valerie M. Weaver, Robert L. Raffai, Matthias Hebrok. nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma [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 A046.

Harnessing endoscopic ultrasound-guided radiofrequency ablation to reshape the pancreatic ductal adenocarcinoma microenvironment and elicit systemic immunomodulation.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognostics and substantial therapeutic challenges, with dismal survival rates. Tumor resistance in PDAC is primarily attributed to its fibrotic, hypoxic, and immune-suppressive tumor microenvironment (TME). Endoscopic ultrasound-guided radiofrequency ablation (EUS-RFA), an Food and Drug Administration (FDA)-approved minimally invasive technique for treating pancreatic cancer, disrupts tumors with heat and induces coagulative necrosis, releasing tumor antigens that may trigger a systemic immune response-the abscopal effect. We aim to elucidate the roles of EUS-RFA-mediated thermal and mechanical stress in enhancing anti-tumor immunity in PDAC. A comprehensive literature review focused on radiofrequency immunomodulation and immunotherapy in pancreatic tumors to understand the pathophysiological mechanisms of RFA and its effect on the TME, which could prevent recurrence and resistance. We reviewed clinical, preclinical, and in vitro studies on RFA mechanisms in pancreatic adenocarcinoma, discussing the unique immunomodulatory effects of EUS-RFA. Recent findings suggest that combining RFA with immune adjuvants enhances responses in pancreatic adenocarcinoma. EUS-RFA offers a dual benefit against PDAC by directly reducing tumor viability and indirectly enhancing anti-tumor immunity. Observations of neutrophil-mediated immunomodulation and programmed cell death ligand 1 (PD-L1) modulation support integrating EUS-RFA with targeted immunotherapies for managing pancreatic adenocarcinoma. Integrating EUS-RFA in PDAC treatment promises direct cytoreduction and synergistic effects with molecular targeted therapies. Prospective clinical trials are crucial to assess the efficacy of this combined approach in improving outcomes and survival rates in advanced PDAC cases.

Deciphering the immune modulation through deep transcriptomic profiling and therapeutic implications of DNA damage repair pattern in hepatocellular carcinoma

AimsDNA damage repair (DDR) plays a pivotal role in hepatocellular carcinoma (HCC), driving oncogenesis, progression, and therapeutic response. However, the mechanisms of DDR mediated immune cells and immuno-modulatory pathways in HCC are yet ill-defined. MethodsOur study introduces an innovative deep machine learning framework for precise DDR assessment, utilizing single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data. Single-cell RNA sequencing data were obtained and in total 85,628 cells of primary or post-immunotherapy cases were analyzed. Large-scale HCC datasets, including 1027 patients in house together with public datasets, were used for 101 machine-learning models and a novel DDR feature was derived at single-cell resolution (DDRscore). Druggable targets were predicted using the reverse phase protein array (RPPA) proteomic profiling of 169 HCC patients and RNA-seq data from 22 liver cancer cell lines. ResultsOur investigation reveals a dynamic interplay of DDR with natural killer cells and B cells in the primary HCC microenvironment, shaping a tumor-promoting immune milieu through metabolic programming. Analysis of HCC post-immunotherapy demonstrates elevated DDR levels that induces epithelial-mesenchymal transition and fibroblast-like transformation, reshaping the fibrotic tumor microenvironment. Conversely, attenuated DDR promotes antigen cross-presentation by dendritic cells and CD8+ T cells, modulating the inflammatory tumor microenvironment. Regulatory network analysis identifies the CXCL10-CXCR3 axis as a key determinant of immunotherapeutic response in low DDR HCC, potentially regulated by transcription factors GATA3, REL, and TBX21. Using machine learning techniques by combining bulk RNA-seq data in house together with public datasets, we introduce DDRscore, a robust consensus DDR scoring system to predict overall survival and resistance to PD-1 therapy in HCC patients. Finally, we identify BRAF as a potential therapeutic target for high DDRscore patients. ConclusionOur comprehensive findings advance our understanding of DDR and the tumor microenvironment in HCC, providing insights into immune regulatory mechanisms mediated via DDR pathways.

Optimized nucleus isolation protocol from frozen mouse tissues for single nucleus RNA sequencing application.

The single cell RNA sequencing technique has been particularly used during the last years, allowing major discoveries. However, the widespread application of this analysis has showed limitations. Indeed, the direct study of fresh tissues is not always feasible, notably in the case of genetically engineered mouse embryo or sensitive tissues whose integrity is affected by classical digestion methods. To overcome these limitations, single nucleus RNA sequencing offers the possibility to work with frozen samples. Thus, single nucleus RNA sequencing can be performed after genotyping-based selection on samples stocked in tissue bank and is applicable to retrospective studies. Therefore, this technique opens the field to a wide range of applications requiring adapted protocols for nucleus isolation according to the tissue considered. Here we developed a protocol of nucleus isolation from frozen murine placenta and pancreas. These two complex tissues were submitted to a combination of enzymatic and manual dissociation before undergoing different steps of washing and centrifugation. The entire protocol was performed with products usually present in a research lab. Before starting the sequencing process, nuclei were sorted by flow cytometry. The results obtained validate the efficiency of this protocol which is easy to set up and does not require the use of commercial kits. This specificity makes it adaptable to different organs and species. The association of this protocol with single nucleus RNA sequencing allows the study of complex samples that resist classical lysis methods due to the presence of fibrotic or fatty tissue, such as fibrotic kidney, tumors, embryonic tissues or fatty pancreas.

Enhancing chemotherapy for pancreatic cancer through efficient and sustained tumor microenvironment remodeling with a fibroblast-targeted nanosystem

Pancreatic cancer (PC) carries a poor prognosis among all malignancies and poses great challenges to clinical drug accessibility due to the severely fibrotic and hypoxic tumor microenvironment (TME). Therein, cancer-associated fibroblasts (CAFs), which are extremely abundant in PC, play a key role in forming the complex PC microenvironment. Therefore, a highly efficient TME reprogramming therapeutic paradigm that can specifically inhibit CAF function is urgently needed. Herein, we successfully developed a novel CAF-tailored nanosystem (Dex-GP-DOCA, DPD) loaded with a potent anti-fibrosis flavonoid compound (Quercetin, QUE), which possesses biological responsiveness to fibroblast activation protein alpha (FAP-α), prolonged TME remodeling and enhancement of clinical chemotherapeutics. Specifically, DPD/QUE allowed for extracellular matrix (ECM) reduction, vessel normalization, hypoxia-induced drug resistance reversal, and blockade of Wnt16 paracrine in CAFs. More importantly, this chemotherapy conducive microenvironment persisted for at least 8 days following treatment with DPD/QUE. It should also be noted that the effective and prolonged microenvironment modulation induced by DPD/QUE significantly improved the chemotherapy sensitivity of Abraxane and gemcitabine, the first-line chemotherapeutic drugs for PC, with inhibition rates increasing from 37.5% and 40.0% to 87.5% and 85.2%, respectively. Overall, our CAFs-targeted nanosystem showed promising prospects for remodeling the TME and facilitating chemotherapy for refractory pancreatic cancer.

Repurposing Tamoxifen for Tumor Microenvironment Priming and Enhanced Tumor-Targeted Drug Delivery.

The dense stromal matrix in fibrotic tumors hinders tumor-targeted drug delivery. Tamoxifen (TMX), an estrogen receptor modulator that is clinically used for the treatment of breast cancer, has been shown to reprogram the tumor microenvironment (TME) and to alleviate desmoplasia. We here investigated if TMX, administered in free and nano-formulated form, can be repurposed as a TME remodeling agent to improve tumor accumulation of nano-formulations in pancreatic ductal adenocarcinoma and triple-negative breast cancer mouse models, evaluated using clinical-stage Cy7-labeled core-crosslinked polymeric micelles (CCPM). Under control conditions, we found higher levels of Cy7-CCPM in PANC-1 tumors (16.7 % ID g-1 at 48 h post i.v. injection) than in 4T1 tumors (11.0 % ID g-1). In both models, free and nano-formulated TMX failed to improve CCPM delivery. These findings were congruent with the results from histopathological immunofluorescence analysis of tumor tissue, which indicated that TMX treatment did not significantly change vascularization, perfusion, macrophage infiltration, collagen density, and collagen fiber thickness. Altogether, our results demonstrate that in PANC-1 and 4T1 mouse models, TMX treatment does not contribute to beneficial TME priming and enhanced tumor-targeted drug delivery.

Hydroxyproline metabolism enhances IFN-γ-induced PD-L1 expression and inhibits autophagic flux

The immune checkpoint protein PD-L1 plays critical roles in both immune system homeostasis and tumor progression. Impaired PD-1/PD-L1 function promotes autoimmunity and PD-L1 expression within tumors promotes immune evasion. If and how changes in metabolism or defined metabolites regulate PD-L1 expression is not fully understood. Here, using a metabolomics activity screening-based approach, we have determined that hydroxyproline (Hyp) significantly and directly enhances adaptive (i.e., IFN-γ-induced) PD-L1 expression in multiple relevant myeloid and cancer cell types. Mechanistic studies reveal that Hyp acts as an inhibitor of autophagic flux, which allows it to regulate this negative feedback mechanism, thereby contributing to its overall effect on PD-L1 expression. Due to its prevalence in fibrotic tumors, these findings suggest that hydroxyproline could contribute to the establishment of an immunosuppressive tumor microenvironment and that Hyp metabolism could be targeted to pharmacologically control PD-L1 expression for the treatment of cancer or autoimmune diseases.

Reversing the PAI-1-induced fibrotic immune exclusion of solid tumor by multivalent CXCR4 antagonistic nano-permeator.

Fibrosis is one of the key factors that lead to the immune exclusion of solid tumors. Although degradation of fiber is a promising strategy, its application was still bottlenecked by the side effects of causing metastasis, resulting in the failure of immunotherapy. Here, we developed an antimetastatic polymer (HPA) for the delivery of chemo-drug and antifibrotic siPAI-1 to form the nano-permeator. Nano-permeator shrank after protonation and deeply penetrated into the tumor core to down-regulate the expression of PAI-1 for antifibrosis, and further promoted the sustained infiltration and activation of T cells for killing tumor cells. Moreover, metastasis after fiber elimination was prevented by multivalent CXCR4 antagonistic HPA to reduce the attraction of CXCL12 secreted by distant organs. The administration of stroma-alleviated immunotherapy increased the infiltration of CD8+ T cells to 52.5% in tumor tissues, inhibiting nearly 90% metastasis by HPA in distant organs. The nano-permeator reveals the mechanism and correlation between antifibrosis and antimetastasis and was believed to be the optimizing immunotherapy for solid fibrotic tumors.

The Association of Fibulin-2 Expression with Histopathological Grade and Fibrotic Tumor Vessel in Meningioma

Background&#x0D; Meningiomas are the most common primary tumors of the central nervous system. About one-fifth of meningiomas tend to recur. The World Health Organization (WHO) histopathological grade was the most useful morphological predictor of recurrence. Fibrotic tumor vessels (FTV) were detected in nearly half of WHO grade I meningiomas and correlated with vascular density and increased risk of recurrence. Fibulin-2 is an extracellular matrix glycoprotein whose expression increases in traumatic central nervous system injuries and can be used as a marker to differentiate the histopathological grade of meningiomas. This study aimed to determine the association of fibulin-2 expression with histopathological grade and FTV in meningiomas.&#x0D; &#x0D; Methods&#x0D; This research was an observational cross-sectional study. The study sample was 36 cases of meningioma in three Anatomical Pathology laboratories in West Sumatra on period January 2019 to December 2020. Samples obtained from resected meningioma cases were then reevaluated on Hematoxylin Eosin (HE) slides to determine the histopathological grade and FTV. Van-Gieson staining was performed to confirm FTV. Fibulin-2 expression in tumor cells was analyzed using immunohistochemical staining. Bivariate statistical analysis using the Chi-Square test with p&lt;0.05 was considered significant.&#x0D; &#x0D; Result&#x0D; High-risk meningiomas (WHO grade II and III) showed high fibulin-2 expression (62.5%), whereas low-risk meningiomas (WHO grade I) were more abundant with low fibulin-2 expression (75%). The presence of FTV at low fibulin-2 expression was 58.3%. Statistical analysis showed no significant association between fibulin-2 expression with histopathological grade (p=0.077) and FTV (p=1,000).&#x0D; &#x0D; Conclusion&#x0D; In summary, the study showed that fibulin-2 expression was not associated to histopathological grade nor to FTV in meningiomas.

Intraoperative Assessment of Breast Cancer Tissues after Breast-Conserving Surgery Based on Mapping the Attenuation Coefficients in 3D Cross-Polarization Optical Coherence Tomography.

Intraoperative differentiation of tumorous from non-tumorous tissue can help in the assessment of resection margins in breast cancer and its response to therapy and, potentially, reduce the incidence of tumor recurrence. In this study, the calculation of the attenuation coefficient and its color-coded 2D distribution was performed for different breast cancer subtypes using spectral-domain CP OCT. A total of 68 freshly excised human breast specimens containing tumorous and surrounding non-tumorous tissues after BCS was studied. Immediately after obtaining structural 3D CP OCT images, en face color-coded attenuation coefficient maps were built in co-(Att(co)) and cross-(Att(cross)) polarization channels using a depth-resolved approach to calculating the values in each A-scan. We determined spatially localized signal attenuation in both channels and reported ranges of attenuation coefficients to five selected breast tissue regions (adipose tissue, non-tumorous fibrous connective tissue, hyalinized tumor stroma, low-density tumor cells in the fibrotic tumor stroma and high-density clusters of tumor cells). The Att(cross) coefficient exhibited a stronger gain contrast of studied tissues compared to the Att(co) coefficient (i.e., conventional attenuation coefficient) and, therefore, allowed improved differentiation of all breast tissue types. It has been shown that color-coded attenuation coefficient maps may be used to detect inter- and intra-tumor heterogeneity of various breast cancer subtypes as well as to assess the effectiveness of therapy. For the first time, the optimal threshold values of the attenuation coefficients to differentiate tumorous from non-tumorous breast tissues were determined. Diagnostic testing values for Att(cross) coefficient were higher for differentiation of tumor cell areas and tumor stroma from non-tumorous fibrous connective tissue: diagnostic accuracy was 91-99%, sensitivity-96-98%, and specificity-87-99%. Att(co) coefficient is more suitable for the differentiation of tumor cell areas from adipose tissue: diagnostic accuracy was 83%, sensitivity-84%, and specificity-84%. Therefore, the present study provides a new diagnostic approach to the differentiation of breast cancer tissue types based on the assessment of the attenuation coefficient from real-time CP OCT data and has the potential to be used for further rapid and accurate intraoperative assessment of the resection margins during BCS.

Evaluation of growth-induced, mechanical stress in solid tumors and spatial association with extracellular matrix content.

Mechanical stresses in solid tumors play an important role in tumor progression and treatment efficacy but their quantification is under-investigated. Here, we developed an experimental and computational approach to calculate growth-induced, residual stresses and applied it to the breast (4T1), pancreatic (PAN02), and fibrosarcoma (MCA205) tumor models. Following resection, tumors are embedded in agarose gels and cuts are made in two perpendicular directions to release residual stress. With the use of image processing, the detailed bulging displacement profile is measured and finite elements models of the bulging geometry are developed for the quantification of the stress levels. The mechanical properties of the tumors are measured in vivo prior to resection with shear wave elastography. We find that the average magnitude of residual stresses ranges from 3.31 to 10.88kPa, and they are non-uniformly distributed within the tissue due to the heterogeneity of the tumor microenvironment. Interestingly, we demonstrate that a second cut can still release a significant amount of stresses. We further find a strong association of spatial hyaluronan and collagen content with the spatial profile of stress for the MCA205 and PAN02 tumors and a partial association for the 4T1. Interestingly the colocalizationof hyaluronan and collagen content had a stronger association with the spatial profile of stress for MCA205, PAN02, and 4T1. Finally, measurements of the elastic modulus with shear wave elastography show a nonlinear correlation with tumor volume for the more fibrotic MCA205 and 4T1 tumors. Overall, our results provide insights for a better understanding of the mechanical behavior of tumors.