Cancer-associated Fibroblasts Cells Research Articles

Development of 42 marker panel for in-depth study of cancer associated fibroblast niches in breast cancer using imaging mass cytometry.

Imaging Mass Cytometry (IMC) is a novel, and formidable high multiplexing imaging method emerging as a promising tool for in-depth studying of tissue architecture and intercellular communications. Several studies have reported various IMC antibody panels mainly focused on studying the immunological landscape of the tumor microenvironment (TME). With this paper, we wanted to address cancer associated fibroblasts (CAFs), a component of the TME very often underrepresented and not emphasized enough in present IMC studies. Therefore, we focused on the development of a comprehensive IMC panel that can be used for a thorough description of the CAF composition of breast cancer TME and for an in-depth study of different CAF niches in relation to both immune and breast cancer cell communication. We established and validated a 42 marker panel using a variety of control tissues and rigorous quantification methods. The final panel contained 6 CAF-associated markers (aSMA, FAP, PDGFRa, PDGFRb, YAP1, pSMAD2). Breast cancer tissues (4 cases of luminal, 5 cases of triple negative breast cancer) and a modified CELESTA pipeline were used to demonstrate the utility of our IMC panel for detailed profiling of different CAF, immune and cancer cell phenotypes.

Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

NNT-AS1 in CAFs-derived exosomes promotes progression and glucose metabolism through miR-889-3p/HIF-1α in pancreatic adenocarcinoma

It is metabolic and signaling crosstalk between stromal cells and tumors in the tumor microenvironment, which influences several aspects of tumor formation and drug resistance, including metabolic reprogramming. Despite considerable findings linking lncRNAs in HIF-1-related regulatory networks to cancer cell, little emphasis has been given to the role in communication between cancer-associated fibroblasts (CAFs) and tumor cells. Previously, we observed that NNT-AS1 was substantially expressed in CAFs cells and CAFs exosomes, and subsequently investigated the influence of CAFs exosomal NNT-AS1 on glucose metabolism, proliferation, and metastasis of pancreatic ductal adenocarcinoma (PDAC) cells. Transmission electron microscopy was used to examine exosomes secreted by PDAC patient-derived CAFs. qRT-PCR was used to evaluate the expression of NNT-AS1, miR-889-3p, and HIF-1. The role of CAFs-derived exosomal NNT-AS1 in PDAC cell progression and metabolism have been identified. Dual luciferase reporter assays examined the binding between NNT-AS1, miR-889-3p, and HIF-1. After PDAC cells co-culture exosomes secreted by CAFs, we found that they alter glucose metabolism, proliferation, and metastasis. In PDAC cells, CAF-derived exosomal lncRNA NNT-AS1 acted as a molecular sponge for miR-889-3p. Furthermore, HIF-1 could be targeted by miR-889-3p and was controlled by NNT-AS1. This study explores the mechanism by which NNT-AS1 influences the interaction of CAFs on glycolytic remodeling, proliferation, and metastasis of tumor cells through regulating miR-889-3p/HIF-1α, which also helps discover new clinical treatment targets for PDAC.

Phenotypic Heterogeneity of Cancer Associated Fibroblasts in Cervical Cancer Progression: FAP as a Central Activation Marker.

Cervical cancer (CC) is the fourth leading cancer among women and is one of the principal gynecological malignancies. In the tumor microenvironment, cancer-associated fibroblasts (CAFs) play a crucial role during malignant progression, exhibiting a variety of heterogeneous phenotypes. CAFs express phenotypic markers like fibroblast activation protein (FAP), vimentin, S100A4, α-smooth muscle actin (αSMA), and functional markers such as MMP9. This study aimed to evaluate the protein expression of vimentin, S100A4, αSMA, FAP, and MMP9 in mesenchymal stem cells (MSC)-CAF cells, as well as in cervical cancer samples. MSC cells were stimulated with HeLa and SiHa tumor cell supernatants, followed by protein evaluation and cytokine profile to confirm differentiation towards a CAF phenotype. In addition, automated immunohistochemistry (IHQa) was performed to evaluate the expression of these proteins in CC samples at different stages. Our findings revealed a high expression of FAP in stimulated MSC cells, accompanied by the secretion of pro/anti-inflammatory cytokines. In the other hand, CC samples were observed to have high expression of FAP, vimentin, αSMA, and MMP9. Most importantly, there was a high expression of their activation proteins αSMA and FAP during the different stages. In the early stages, a myofibroblast-like phenotype (CAFs αSMA+ FAP+), and in the late stages a protumoral phenotype (CAF αSMA- FAP+). In summary, FAP has a crucial role in the activation of CAFs during cervical cancer progression.

Abstract 218: Deciphering tumoroid-CAF interactions through a spatially segregated coculture model

Abstract In the context of lung cancers, cancer-associated fibroblast (CAF) plays a pivotal role as a key component of the tumor microenvironment (TME), affecting tumor growth, invasion, metastasis, immune modulation, and drug resistance. Despite the recognized impact of CAFs, the precise interaction between the CAF and tumor cell remains elusive. Previous studies have reported that CAFs stimulate tumor cell growth both in vitro and in vivo. However, our tumoroid models reveal an intriguing phenomenon where CAFs inhibit tumoroids growth within Matrigel. This led us to hypothesize that paracrine signaling might be the primary mode of interaction between CAFs and tumor cells. To unravel the intricacies of this interaction, we developed an innovative coculture method. Using hydrophobic barrier, we segregated the coculture of nine sets of CAFs and tumoroids within a single well, preventing CAFs from infiltrating the tumoroid culture area. This setup enabled interaction solely through paracrine signaling. Surprisingly, cocultured tumoroids exhibited no significant differences compared to individually cultured tumoroids. Conversely, cocultured CAFs displayed a remarkable increase in growth compared to their individually cultured counterparts. This suggests that tumoroids influence CAF growth, while CAFs do not significantly impact tumoroid growth. Furthermore, in three coculture sets, tumor cells within the Matrigel migrated towards the CAF culture area, indicating that CAFs induce tumor cell metastasis. Immunofluorescence staining confirmed this metastatic phenomenon. To identify the paracrine factors influencing CAFs and tumoroids, we employed RNA-sequencing, single-cell RNA-sequencing, and proteomic analysis of culture soup. Proteomic analysis revealed a substantial increase in proteins and signaling pathways related to the extracellular matrix in cocultures compared to single cultures. Our findings suggest that tumor cells recruit CAFs, promoting CAF growth to establish and fortify their TME, ultimately leading to drug resistance, metastasis, and immune modulation Citation Format: Sung Min Kim, Yoo Ri Ko, Hye Seon Park, Se Jin Jang. Deciphering tumoroid-CAF interactions through a spatially segregated coculture model [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 218.

Abstract 3878: Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis

Abstract The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) is driven by cell-intrinsic features and cell-extrinsic interactions between tumor cells and the desmoplastic stroma, which is infiltrated with heterogeneous populations of cancer-associated-fibroblasts (CAFs) and immune cells. These features are able to drive emergent properties such as chemoresistance through diverse and not yet fully elucidated mechanisms. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of PDAC patient tumors, we previously reported marked heterogeneity in the malignant cells and CAF composition of PDAC. In this project, we performed high-plex single-cell spatial molecular imaging (990-plex SMI, Nanostring) to dissect the therapy-remodeled multicellular neighborhoods and malignant-stroma cell interactions in primary resected human pancreatic cancer with (n = 6) or without (n = 7) neoadjuvant chemotherapy and radiotherapy. SMI of the PDAC samples yielded ~753,000 high-confidence single cells and captured the heterogeneity of malignant cells and CAFs. We identified three malignant subtypes: classical (CLS), basal-like (BSL), and the recently characterized neural-like progenitor (NRP) malignant subtype that exhibits stem-like and neurodevelopmental features. To leverage the single-cell spatial resolution of SMI to further dissect treatment-associated changes in cell-cell interactions, we developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked enrichment in ligand-receptor interactions between CAFs and malignant cells in response to treatment. In combination with several complementary methods, including examination of downstream target gene expression and ex vivo 3D co-culture of CAF and PDAC cells, we found that IL-6 family signaling was the most consistently enriched interaction between CAFs and malignant cells after treatment. This led us to hypothesize that IL-6 signaling between CAFs and malignant cells may induce chemoresistance in cancer cells. To test this hypothesis, we treated three human cancer cell lines (AsPC1, Panc1, Panc0203) with 5FU with or without IL-6 and discovered that IL-6 induced chemoresistance for two of the lines (Panc1, Panc0203). Taken together, we have integrated novel experimental and computational approaches to enable high-resolution, spatially-guided discovery of treatment-associated remodeling in the pancreatic cancer microenvironment. Further studies will be conducted to dissect additional mechanisms by which candidate cell-cell interactions promote disease progression and/or resistance to cytotoxic therapy, with the ultimate goal of guiding novel therapeutic development. Citation Format: JINGYI CAO, Carina Shiau, Dennis Gong, Mark T. Gregory, Xunqin Yin, Jae-Won Cho, Peter L. Wang, Jennifer Su, Jason W. Reeves, Jimmy A. Guo, Nicole A. Lester, Jung Woo Bae, Ryan Zhao, Martin Hemberg, William L. Hwang. Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis [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 3878.

Abstract 5500: Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain metastasis

Abstract Introduction: Brain metastasis (BM) remains incurable with a dismal 2-year survival of less than 2%. BM are surrounded by a complex tumor microenvironment (TME) comprised of a meshwork of extracellular matrix (ECM) proteins and an assemblage of cell types including cancer-associated fibroblasts (CAFs), the most prominent cell type within the TME. We have previously shown that patient-derived BM CAFs can either support or inhibit tumor growth in vivo, suggesting that BM CAFs are heterogenous. Yet, there remains a dearth of data on the functional and mechanistic underpinnings of CAFs in the BM TME. Methods: To explore both the molecular profiles of CAFs and their heterogeneity in the BM TME, we performed a single cell Multiome (RNA and Assay for Transposase-Accessible Chromatin (ATAC)) sequencing (scMultiome-seq) analysis of 9 human BM tissue samples and 4 BM patient-derived CAF cell lines, from both lung and breast primary cancers. Next, we investigated the tumor supportive or inhibitory capabilities of BM CAF cell lines on the growth of BM tumor cells in vitro. Results: ScMultiome-seq analysis of BM tissue samples identified 4 distinct CAF subpopulations, that we termed desmoplastic, immune, contractile, and neural CAFs, in the BM TME. These CAF subpopulations are respectively involved in either regulating the ECM organization, orchestrating the neuroimmune response, triggering tissue contraction or regulating neural components upon BM growth. CellPhoneDB analysis revealed that CAFs had more cell-cell interactions with other cells than any other cell types in the BM TME. Pseudotime analysis demonstrated the potential cell of origin of each BM CAF subpopulation and the cell state transition they undergo. We observed that the end cell state of the desmoplastic CAFs expressed high levels of immunoglobulin superfamily containing leucine rich repeat (ISLR). ISLR was also observed to be highly expressed in a BM patient-derived CAF cell line which was previously reported by our lab to restrain tumor growth in vivo, suggesting that desmoplastic CAFs with high ISLR expression (ISLR-CAFs) might be tumor inhibitory. ScMultiome-seq analysis of BM CAF cell lines validated that tumor inhibitory CAF cell lines were primarily of the desmoplastic ISLR-CAF subpopulation. Conditioned media obtained from ISLR-CAFs containing high levels of ISLR inhibited tumor cell viability in vitro compared to controls. Conclusion: Our study is the first to describe the heterogeneity of CAFs and their potential central roles in the BM TME. We also identify that CAF subpopulations are dynamic and can transition to tumor inhibitory cell states, such as ISLR-CAFs. These data have potential to help design therapeutic strategies that selectively target tumor supportive CAFs whilst protecting tumor inhibitory ones. Citation Format: Thomas Simon, David N. Buckley, Ben Y. Tew, Matthew Salomon, Zeyi Yang, Gerald C. Gooden, Steven A. Toms, Gabriel Zada, Bodour Salhia. Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain metastasis [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 5500.

CLCN3 in mediating the proliferation of human ovarian cancer cells.

Chloride channel-3 (CLCN3), a crucial component of the voltage-gated chloride channel family, is implicated in numerous physiological and pathophysiological processes. This study aimed to investigate the characteristics of CLCN3 in pancancer and its influence on the immune response through the use of a range of databases. Concurrently, we assessed the impact of CLCN3 on the proliferation of ovarian cancer (OC) cells and explored its potential mechanisms. We employed the Tumor Immune Estimation Resource (TIMER) 2.0 and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to examine the messenger RNA (mRNA) and the protein expression of CLCN3 across various cancers. The prognostic significance of CLCN3 was evaluated using the Gene Expression Profiling Interactive Analysis 2.0 (GEPIA 2.0) database. The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) facilitated the analysis of CLCN3 promoter methylation levels. The association between CLCN3 expression and tumor-infiltrating immune cells was investigated using various algorithms. The cBioportal database facilitated the analysis of CLCN3 mutations and mutation sites across various cancers. The Tumor-Immune System Interactions Database (TISIDB) database was employed to explore the correlation between CLCN3 expression and immune or molecular subtypes across a variety of cancer types. We collected ovarian tissue samples, encompassing both normal ovarian and OC tissues. The human OC cell lines, SKOV3 cells and OVCAR433 cells, were cultured. CLCN3 expression was determined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR), while phosphatidylinositol 3-kinase/Akt kinase (PI3K/AKT) expression was detected using Western blot. We utilized small interfering RNA (siRNA) technology to suppress CLCN3 expression. The proliferative capacity of SKOV3 and OVCAR433 cells was assessed using the Cell Counting Kit 8 (CCK-8) assay. CLCN3 demonstrated an aberrant expression in a number of cancer types and was markedly reduced in OC tissues. Poor prognosis in cervical squamous cell cancer and myeloid leukemia was linked to excessive expression of CLCN3. The examination of immune cell infiltration, which included CD8+ T cells, B cells, T regulatory cells, and cancer-associated fibroblast cells, showed a strong association with aberrant CLCN3 expression. Following the use of siRNA technology, the ability of the ovarian carcinoma cell line SKOV3 and OVCAR433 to proliferate as well as the expression of PI3K/AKT both increased. CLCN3 is a possible biomarker for immune-related processes and the prognosis of cancer, and the PI3K/AKT signaling pathway may affect OC cells' ability to proliferate.

Viscoelastic stiffening of gelatin hydrogels for dynamic culture of pancreatic cancer spheroids

The tumor microenvironment (TME) in pancreatic adenocarcinoma (PDAC) is a complex milieu of cellular and non-cellular components. Pancreatic cancer cells (PCC) and cancer-associated fibroblasts (CAF) are two major cell types in PDAC TME, whereas the non-cellular components are enriched with extracellular matrices (ECM) that contribute to high stiffness and fast stress-relaxation. Previous studies have suggested that higher matrix rigidity promoted aggressive phenotypes of tumors, including PDAC. However, the effects of dynamic viscoelastic matrix properties on cancer cell fate remain largely unexplored. The focus of this work was to understand the effects of such dynamic matrix properties on PDAC cell behaviors, particularly in the context of PCC/CAF co-culture. To this end, we engineered gelatin-norbornene (GelNB) based hydrogels with a built-in mechanism for simultaneously increasing matrix elastic modulus and viscoelasticity. Two GelNB-based macromers, namely GelNB-hydroxyphenylacetic acid (GelNB-HPA) and GelNB-boronic acid (GelNB-BA), were modularly mixed and crosslinked with 4-arm poly(ethylene glycol)-thiol (PEG4SH) to form elastic hydrogels. Treating the hybrid hydrogels with tyrosinase not only increased the elastic moduli of the gels (due to HPA dimerization) but also concurrently produced 1,2-diols that formed reversible boronic acid-diol bonding with the BA groups on GelNB-BA. We employed patient-derived CAF and a PCC cell line COLO-357 to demonstrate the effect of increasing matrix stiffness and viscoelasticity on CAF and PCC cell fate. Our results indicated that in the stiffened environment, PCC underwent epithelial-mesenchymal transition. In the co-culture PCC and CAF spheroid, CAF enhanced PCC spreading and stimulated collagen 1 production. Through mRNA-sequencing, we further showed that stiffened matrices, regardless of the degree of stress-relaxation, heightened the malignant phenotype of PDAC cells. Statement of significanceThe pancreatic cancer microenvironment is a complex milieu composed of various cell types and extracellular matrices. It has been suggested that stiffer matrices could promote aggressive behavior in pancreatic cancer, but the effect of dynamic stiffening and matrix stress-relaxation on cancer cell fate remains largely undefined. This study aimed to explore the impact of dynamic changes in matrix viscoelasticity on pancreatic ductal adenocarcinoma (PDAC) cell behavior by developing a hydrogel system capable of simultaneously increasing stiffness and stress-relaxation on demand. This is achieved by crosslinking two gelatin-based macromers through orthogonal thiol-norbornene photochemistry and post-gelation stiffening with mushroom tyrosinase. The results revealed that higher matrix stiffness, regardless of the degree of stress relaxation, exacerbated the malignant characteristics of PDAC cells.

IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts

BackgroundIt remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma.MethodsRNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs.ResultsIn this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner.ConclusionsWe disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

Association of α-SMA and EpCAM Expressions with Recurrence Risk Based on Histopathological Subtypes of Basal Cell Carcinoma

Background Basal cell carcinoma (BCC) is the most common skin malignancy in the world with a proportion 70%. Recurrent and aggressive variants are still challenging in diagnosis and treatment. Histopathologically, there are two group of BCC risk of recurrence, i.e low risk and high risk. Assessment the expression of  alpha-smooth muscle actin (α-SMA) as a biomarker of cancer associated fibroblast cell in stroma and the expression of epithelial cell adhesion molecule (EpCAM) in tumor cells probably have role in the pathomechanism of BCC progression, suggest its can distinguish the risk of BCC recurrence. This research aims to determine the assosiation between the expression of α-SMA and EpCAM with the recurrence risk group of BCC histopathological subtypes.   Methods This was a cross sectional study using 48 samples, with 24 low and high risk groups each. Histopathological subtypes were determined from Hematoxylin and eosin slide. The expression of α-SMA and EpCAM was examined by the immunohistochemical method which was assessed semiquantitatively. Statistical analysis was performed using the Chi-square test with p<0.05  was considered significant.   Results Expression of α-SMA with the score 3 was found more in high risk BCC (85.7%), while a score of 1 was more in low risk BCC (100%). Loss of EpCAM expression was mainly found in high risk BCC (82.8%). Statistical analysis showed that there was a significant assosiation between the expression of α-SMA and EpCAM and the recurrence risk group of BCC histopathological subtypes with p value =0.000 for each.   Conclusions This study concluded that increased of α-SMA expression and loss of EpCAM expression were associated with a high recurrence risk group of BCC histopathological subtypes.    

Spatiotemporally controlled Pseudomonas exotoxin transgene system combined with multifunctional nanoparticles for breast cancer antimetastatic therapy

The tumor microenvironment is a barrier to breast cancer therapy. Cancer-associated fibroblast cells (CAFs) can support tumor proliferation, metastasis, and drug resistance by secreting various cytokines and growth factors. Abnormal angiogenesis provides sufficient nutrients for tumor proliferation. Considering that CAFs express the sigma receptor (which recognizes anisamide, AA), we developed a CAFs and breast cancer cells dual-targeting nano drug delivery system to transport the LightOn gene express system, a spatiotemporal controlled gene expression consisting of a light-sensitive transcription factor and a specific minimal promoter. We adopted RGD (Arg-Gly-Asp) to selectively bind to the αvβ3 integrin on activated vascular endothelial cells and tumor cells. After the LightOn system has reached the tumor site, LightOn gene express system can spatiotemporal controllably express toxic Pseudomonas exotoxin An under blue light irradiation. The LightOn gene express system, combined with multifunctional nanoparticles, achieved high targeting delivery efficiency both in vitro and in vivo. It also displayed strong tumor and CAFs inhibition, anti-angiogenesis ability and anti-metastasis ability, with good safety. Moreover, it improved survival rate, survival time, and lung metastasis rate in a mouse breast cancer model. This study proves the efficacy of combining the LightOn system with targeted multifunctional nanoparticles in tumor and anti-metastatic therapy and provides new insights into tumor microenvironment regulation.

Abstract C015: Clinically usable classification of cancer-associated fibroblast subtypes in pancreatic cancer

Abstract Background: Cancer-associated fibroblasts (CAFs) in pancreatic adenocarcinoma (PDAC) are known to play a significant role in regulating tumor progression, invasion, and metastasis. Multiple studies using experimental strategies, as well as single-cell RNA-sequencing (scRNAseq) technology, have shown the existence of subpopulations of PDAC CAF cells, with divergent physical and biological characteristics. However, how to translate CAF cell subpopulations to the clinic remains unclear. Methods: We developed a computational method for scRNAseq, SCISSORS, to identify rare cell clusters and determine exemplar genes. Using SCISSORS, we have defined human exemplar genes of the previously described myCAF and iCAF cell subpopulations using scRNAseq data. These exemplar genes were then used to develop a single-sample classifier (SSC) for PDAC CAFs that we call CAPER in bulk RNAseq data. CAPER was trained using penalized logistic regression in 4 datasets (n= 372) and validated in 8 datasets (n= 593). Results: SCISSORS was able to identify rare cells such as previously described apCAFs (0.59% of cells) and basal-like cell clusters (0.092% of cells) that were missed in the previous analysis. We hypothesized that the sensitivity of SCISSORS would allow us to define exemplar genes that may be applied to bulkRNAseq. Indeed, the top ranked 25 exemplar genes defined by SCISSORS were associated with overall survival (OS) (10 datasets, n=948, p=0.017, log-rank test). To develop a clinically usable classifier, we used the SCISSORS exemplar genes to develop the SSC CAPER which consists of 9 gene pairs. CAPER CAF calls in the validation datasets showed high reproducibility compared with cluster-based labels (AUC: 0.956). In a meta-analysis, patients with CAPER myCAF subtypes showed shorter OS (median OS: 18.0m) compared to CAPER iCAF subtypes (median OS: 28.7m) (p<0.001, log-rank test). Multivariable analysis found that CAPER CAF subtypes were independently prognostic when included with PurIST tumor subtype calls (CAPER p<0.001, PurIST p<0.001, Cox regression). Interestingly, patients subtyped as basal-like for the tumor and myCAF for the CAF showed the worst outcome with a median OS of 11.2 months; while patients subtyped as classical for the tumor and iCAF for the CAF showed the best outcome with a median OS of 30.2 months (p<0.001, log-rank test). Conclusions: CAF genes have largely been evaluated in scRNAseq. We have defined CAF exemplar genes that can be applied to bulk RNAseq, and have developed a clinically usable classifier similar to the now CLIA-approved PurIST that we call CAPER that is robust and accurate for the classification of CAF subtypes in patients. The ability to evaluate CAF subtypes in bulk gene expression provides a powerful tool that may be applied to the plethora of data from patients and clinical trials. CAF subtypes are independently prognostic compared to tumor-intrinsic subtypes, supporting important biological implications, and may facilitate the design of future clinical trials. Citation Format: Xianlu L. Peng, Elena Kharitonova, Joseph Kearney, Ian McCabe, Madison Jenner, Ashley B. Morrison, Alina Iuga, Naim U. Rashid, Jen Jen Yeh. Clinically usable classification of cancer-associated fibroblast subtypes 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 C015.

Abstract A042: Cancer associated fibroblasts drive transcriptional changes in tumor cells from classical to basal phenotype and promote epithelial-to-mesenchymal transition in human pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous tumor comprised of epithelial tumor, endothelial, immune, and importantly, cancer associated fibroblasts (CAFs) cells. CAFs drive a complex tumor microenvironment (TME) through mechanisms of intratumoral interactions that are incompletely understood. It’s crucial to account for the complex role of CAFs in modern ex-vivo tumor systems as regulators of both promoting and restraining tumor growth, providing growth factor support, reprogramming immune cells, and altering the TME. Using our patient-derived organoid (PDO) and CAF coculture, we identify novel intercellular interactions between CAF and PDO that promote tumor cell proliferation, impair response to therapy, and alter transcriptional phenotype. Methods: We interrogate mechanisms of cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture of PDO and CAFs, established from patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing-based transcriptomics, comprehensive proteome profiling for 107 secreted proteins with curated validation by ELISA, cellular phenotyping by multiparameter flow cytometry, and qPCR for validation of curated gene lists. Results: Bulk RNAseq of FACS sorted CAF – PDO cocultures from 12 patients demonstrate Moffitt classification transcriptional changes in 42% of samples compared to PDO monoculture, suggesting CAFs drive basal tumor phenotype. To investigate protein level changes, we compared the secreted proteome of PDOs that transitioned to a basal phenotype after coculture to PDOs that did not undergo transition. Proteome analysis from coculture supernatant revealed that HGF, GDF15, TFF3, and VEGF-A are significantly increased in samples that undergo classical to basal transition. Pathway analysis comparing transcriptional data from classical and basal PDOs identifies significant upregulation of pathways associated with epithelial-to-mesenchymal transition (EMT), inflammation, and NF-kB in basal PDOs. Further, coculture leads to decreased expression of E-cadherin and increased expression of N-cadherin on the surface of PDOs while CAF markers remain unchanged. This interaction is accompanied by an increase in PDO proliferation in coculture, suggesting that CAF presence enhances tumorgenicity and changes epithelial tumor cell fitness. Ongoing experiments will investigate tumor cell-CAF interactions spatially using matched patient tissue to complement the in vitro coculture system. Conclusions: Interactions between cancer cells and CAFs drive overall tumor biology via a complex TME and contribute to poor PDAC patient outcomes. Using our patient matched PDO - CAF coculture, we demonstrate that a CAF competent TME drives PDO plasticity towards a more basal transcriptional phenotype and enhanced EMT. We introduce an elegant approach combining in vitro coculture experiments and high dimensional assays to investigate the complex biology of the TME and inform the mechanisms driving cancer biology in individual patients with PDAC. Citation Format: Samantha Guinn, Joseph Tandurella, Jae W. Lee, Daniel J. Zabransky, Mili Ramani, Jignasha Patel, Elana J. Fertig, Elizabeth M. Jaffee, Richard A. Burkhart, Jacquelyn W. Zimmerman. Cancer associated fibroblasts drive transcriptional changes in tumor cells from classical to basal phenotype and promote epithelial-to-mesenchymal transition in human pancreatic ductal adenocarcinoma [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 A042.

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

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a dense stroma primarily composed of cancer associated fibroblasts (CAFs). Our group recently performed a lineage tracing study using genetically engineered mouse models. This study demonstrated that CAFs and pancreatic tissue resident fibroblasts (TRFs) are both derived from the fetal splanchnic mesenchyme, an area of mesoderm next to the foregut endoderm. To determine whether two major subtypes of CAFs are derived from the splanchnic mesenchyme, I analyzed co-expression of the lineage tracing marker Tomato and CAF subtype marker a smooth muscle actin (aSMA). This suggests that both myofibroblasts (aSMA high) and inflammatory fibroblasts (aSMA low) are derived from the splanchnic mesenchyme. Furthermore, we found that transcription factor Gata6 is expressed in the splanchnic mesenchyme and in some CAFs. We constructed mouse genetic models where Gata6 is specifically deleted in fibroblasts. These Gata6 conditional knock out mice had increased tumor burden, suggesting that stromally-expressed Gata6 has a tumor restraining role. We derived CAF and TRF cell lines from these genetic mouse models and their littermate normal mice. My preliminary data suggest that several important fibroblast factors, including type I collagen, aSMA, and Interleukin 6, are upregulated in the Gata6 KO CAFs and TRFs relative to normal CAFs and TRFs. In summary, our study showed that two major subtypes of pancreatic CAFs are derived from the splanchnic mesenchyme, and splanchnic factors may play a role in adult pancreatic fibroblasts to regulate the expression of critical fibroblast genes. Citation Format: Tom Walter, Lu Han, Michael Ostrowski. The involvement of the splanchnic program at the cellular and molecular levels in pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C032.

Abstract B062: CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to therapy. Stromal cancer-associated fibroblasts (CAFs) play a key role in promoting tumor progression and chemoresistance, by creating a fibrotic microenvironment that impedes drug access and feeds PDAC cells nutrients and pro-tumor signals. CRO-67 (Patent PCT/AU2023/050505) is a novel drug developed with Noxopharm Ltd using a rational medicinal chemistry design to improve bioavailability of chromans which have potent anti-cancer activity. Using our patient-derived PDAC tumor in a dish model (explants; maintain multicellular architecture and fibrosis) we previously showed that CRO-67 has both anti-tumor and CAF reprogramming capacity in 4 patient explants [Cancer Res (2022) 82 (22_Supplement):C073]. Aims: 1) To expand evaluation of CRO-67 in additional patient-derived PDAC tumor explants given PDAC heterogeneity. 2) Assess the effect of CRO-67 on PDAC cell and CAF function. 3) Validate the therapeutic potential of CRO-67 on PDAC growth in vivo. Methods: 1) PDAC tumor samples collected from 7 patients undergoing pancreatic resection. Tumor explants (1–2mm diameter) were cultured on gelatin sponges, treated with CRO-67 (0–50μg/mL) every 3 days and fixed on day 12. Therapeutic response assessed by immunohistochemistry for cytokeratin (PDAC cells), α-smooth muscle actin (CAFs), bromodeoxyuridine (proliferation) and TUNEL (cell death). 2) PDAC cells (MiaPaCa-2) and patient-derived CAFs were treated with CRO-67 (1.5μM) for 48h before proliferation analysis (IncuCYTE S3) and for 24h before apoptosis (Annexin V/DAPI staining; flow cytometry) and cell cycle (DAPI staining; flow cytometry) analysis. 3) Subcutaneous PDAC (BxPC3 human cells) tumors in mice were treated with CRO-67 (2.5mg/kg Intraperitoneally, twice a day) for 21 days and tumor volume measured (calipers). Results: 1) CRO-67 treatment decreased tumor and CAF cell frequency in 6/6 (no quantifiable tumor in patient 7 explants) and 7/7 patient PDAC explants, respectively. It also decreased explant cell proliferation and increased cell death versus controls. 2) CRO-67 completely abolished proliferation of MiaPaCa-2 and reduced the average growth rate of CAFs by 81.3±8.8% (p=0.0025; n=5), increased apoptosis in MiaPaCa-2 by 455.7±21.4% (p=0.003; n=3) and in CAFs by 172.6±13.1% (p=0.0067; n=5) and increased the fraction of cells in G2/M cell cycle phase by 163.8±12.8% (p<0.0001; n=3) in MiaPaCa-2 and by 74.6±8.6% (p=0.0015; n=5) in CAFs versus controls. 3) CRO-67 reduced PDAC tumors in vivo by 56.7±6.6% (p=0.0013; n=9 mice/group) versus controls. Conclusions: CRO-67: 1) reduced PDAC cells and CAFs in human PDAC explants via reduced proliferation and increased death; 2) reduced proliferation of PDAC cells and CAFs in vitro, increased apoptosis and disrupted cell cycle progression through G2/M phase; 3) reduced PDAC tumor growth in vivo. Implication: We predict CRO-67 is a potential ‘dual cell’ therapy with direct anti-tumor effects and CAF reprogramming capacity, warranting further investigation in vivo. Citation Format: Keilah Garcia Netto, Shannon Chiang, John Kokkinos, Koroush S. Haghighi, Aparna Raina, Omali Pitiyarachchi, Janet Youkhana, Quach Truong, Daniel Wenholz, Xiang Li, Olivier Laczka, Naresh Kumar, John Wilkinson, David Goldstein, George Sharbeen, Phoebe A. Phillips. CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B062.

Abstract A021: Adjuvant iC9.B7-H3 CAR T cell-based immunotherapy effectively eradicates local and distant metastases in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Despite negative margin resections of the cancer and systemic therapy, PDAC often recurs. Pancreatic cancer-associated fibroblasts (CAFs) contribute to a highly desmoplastic tumor microenvironment (TME), preventing T cell infiltration. Moreover, CAF-secreted factors suppress the anti-tumor activity of T cells and shift tumor cells toward invasive proliferative and mesenchymal phenotypes. Therefore, targeting CAFs can enhance T-cell infiltration and improve treatment outcomes. In this study, we investigate using Chimeric Antigen Receptor (CAR) T cells targeting B7-H3 to eliminate PDAC metastatic disease in a preclinical mouse model. B7-H3, an immune checkpoint molecule, is highly expressed on PDAC cells and CAFs. Our CART-B7-H3 construct includes an inducible caspase 9 (iC9) suicide gene, which allows for the controlled elimination of the CAR T cells in the event of overwhelming cytokine release syndrome. Methods: The efficacy of iC9.B7-H3 CAR T cells was tested in vitro against human PDAC and fibroblast cell lines at various ratios. The antitumor activity of iC9.B7-H3 CAR T cells was assessed in PDAC cells after exposure to CAF-conditioned media, simulating TME with immunosuppressive cytokines. To validate the therapeutic potential of iC9.B7-H3 CAR T cells in vivo, NSG mice were orthotopically engrafted with patient-derived PDAC6 cells and hCAF1 cells at a ratio of 1:9, reflecting a clinically relevant model. After three weeks, the mice underwent surgical resection of the primary tumor (distal pancreatectomy and splenectomy) and were subsequently treated systemically with iC9.B7-H3 CAR T cells. Results: In vitro, at a high effector-to-target (E:T) ratio, iC9.B7-H3 CAR T cells effectively eliminated PDAC cells and CAFs. However, their anti-tumor activity was diminished when the E:T ratio was lowered (PDAC6 tumor cell killing (TCK): 92±5% at 1:1, hCAF1 TCK: 81.67±5% at 1:1, compared to PDAC6 TCK: 26±5% at 1:10, hCAF1 TCK: 0% at 1:10, P<0.0001). PDAC cells co-cultured with CAF cells demonstrated reduced susceptibility to elimination by iC9.B7-H3 CAR T cells compared to PDAC cells alone (PDAC3 TCK: 92±5% at 1:1, PDAC3+hCAF1 (1:9 ratio) TCK: 81±5% at 1:1, P=0.002). However, incubation with CAF-conditioned media did not significantly affect the elimination of PDAC cells by iC9.B7-H3 CAR T cells, which may suggest that CAFs may require direct cell-cell contact to exert their effects. In vivo, iC9.B7-H3 CAR T cells effectively eradicated both local and distant PDAC metastases for the length of the experiment (160 days), indicating long-term efficacy in the treatment of PDAC metastases with no adverse side effects. Conclusion: These data suggest that iC9.B7-H3 CAR T cells may be an effective adjuvant treatment for the eradication of micro-metastases and the prevention of disease recurrence in PDAC patients. Citation Format: Shahrzad Arya, Seyed Amir Sanatkar, Cristina Martin, Marco Ventin, Jingyu Jia, Giulia Cattaneo, Gabriella Lionetto, David T. Ting, Xinhui Wang, Sandra Ryeom, Cristina R. Ferrone, Soldano Ferrone. Adjuvant iC9.B7-H3 CAR T cell-based immunotherapy effectively eradicates local and distant metastases in pancreatic ductal adenocarcinoma [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 A021.

Knockout of Shcbp1 sensitizes immunotherapy by regulating α-SMA positive cancer-associated fibroblasts.

The crucial role of cancer-associated fibroblasts (CAFs) in promoting T-cell exclusion has a significant impact on tumor immune evasion and resistance to immunotherapy. Therefore, enhancing T-cell infiltration into solid tumors has emerged as a pivotal area of research. We achieved a conventional knockout of Shcbp1 (Shcbp1-/- ) through CRISPR/Cas9 gene editing and crossed these mice with spontaneous breast cancer MMTV-PyMT mice, resulting in PyMT Shcbp1-/- mice. The different CAF subtypes were detected by flow cytometry analysis (FCA). We evaluated collagen and CAFs levels using Sirius red staining, immunohistochemistry (IHC), and immunofluorescence (IF). Primary tumor cells and CAFs were isolated from both PyMT Shcbp1+/+ and PyMT Shcbp1-/- mice. We analyzed CAFs' proliferation, invasion, migration, apoptosis, and cell cycle. Transwell coculture experiments were performed with primary tumor cells and CAFs to evaluate the role of CAFs in increasing the sensitivity of tumor cells to Erdafitinib. Tumors from PyMT Shcbp1+/+ and PyMT Shcbp1-/- mice were orthotopically transplanted to assess the therapeutic effect of the Erdafitinib and PD-1 combination. CAFs and T-cell infiltration in these tumors were assessed using FCA and IF. Knockout of Shcbp1 leads to a significant reduction in tumor burden, promotes longer survival, and decreases CAFs in MMTV-PyMT. Moreover, knockout of Shcbp1 enhances the sensitivity of Erdafitinib, leading to effective inhibition of CAFs' proliferation and invasion, as well as the induction of apoptosis. Additionally, it results in cell cycle arrest at the G2/M phase in vitro. Meanwhile, Shcbp1-/- CAFs change the sensitivity of Shcbp1-/- tumor cells to Erdafitinib compared to Shcbp1+/+ CAFs. Importantly, knockout of Shcbp1 boosts the effectiveness of Erdafitinib in combination with immune checkpoint blockade therapyby augmenting T-cell infiltration through CAFsregulation in vivo. Our findings demonstrate that knockout of Shcbp1 holds significant potential in enhancing the therapeutic response of Erdafitinib combined with PD-1 antibody treatment, offering promising prospects for future breast cancer therapies.

Bioengineered hydrogels enhance ex vivo preservation of patient-derived tumor explants for drug evaluation

Ex vivo patient-derived tumor slices (PDTS) are currently limited by short-term viability in culture. Here, we show how bioengineered hydrogels enable the identification of key matrix parameters that significantly enhance PDTS viability compared to conventional culture systems. As demonstrated using single-cell RNA sequencing and high-dimensional flow cytometry, hydrogel-embedded PDTS tightly preserved cancer, cancer-associated fibroblast, and various immune cell populations and subpopulations in the corresponding original tumor. Cell-cell communication networks within the tumor microenvironment, including immune checkpoint ligand-receptor interactions, were also maintained. Remarkably, our results from a co-clinical trial suggest hydrogel-embedded PDTS may predict sensitivity to immune checkpoint inhibitors (ICIs) in head and neck cancer patients. Further, we show how these longer term-cultured tumor explants uniquely enable the sampling and detection of temporal evolution in molecular readouts when treated with ICIs. By preserving the compositional heterogeneity and complexity of patient tumors, hydrogel-embedded PDTS provide a valuable tool to facilitate experiments targeting the tumor microenvironment.

Multidimensional progressive single-cell sequencing reveals cell microenvironment composition and cancer heterogeneity in lung cancer.

Despite substantial advances in cancer biology and treatment, the clinical outcomes of patients with lung cancer remain unsatisfactory. The tumor microenvironment (TME) is a potential target. Using single-cell RNA sequencing, we could distinguish eight distinct cell types in the lung cancer microenvironment, demonstrating substantial intratumoral heterogeneity in 19 different lung cancer tumor samples. Through the re-dimensional grouping of cancer-associated fibroblasts (CAFs), myeloid cells, epithelial cells, natural killer (NK) cells, and T cells, the difference in the TME of lung cancer was revealed. We discovered SFTPB, SFN, and KRT8 as possible predictive biomarkers for lung cancer by assessing the gene expression patterns in epithelial cells. Examining cell-to-cell communications showed a robust association between the quantity of matrix CAFs, epithelial cells, and macrophages in the thrombospondin signaling pathway. Additionally, we found that the amyloid precursor protein signaling pathway primarily originated from the matrix, and inflammatory cancer-associated endothelial and fibroblast cells showed a co-expression relationship with myeloid cells and B cells. Through cell-to-cell correlation analysis, we found positive regulation between NK cells, regulatory T cells, GZMB-CD8 T cells, and GZMK-CD8 T cells, which could play a role in developing immune TMEs. These findings support studies on cancer heterogeneity and add to our understanding of lung cancer's cellular microenvironment.