Patient-derived 3D Organoids Research Articles

The anticancer effects of Aronia berry extract are mediated by Chk1 and p53 in colorectal cancer

BackgroundAronia berry extracts (ABE) have recently been reported to possess significant anti-cancer effects in various malignancies, including colorectal cancer (CRC), due to their high polyphenolic content. However, the molecular mechanism(s) underlying the anti-cancer effects of ABE in CRC remain unclear, which is important to consider when considering their use as complementary medicine approaches in cancer. MethodsWe performed genome-wide transcriptomic profiling and pathway enrichment analysis to identify specific growth signaling pathways associated with ABE treatment in CRC cells. In addition, a series of systematic and comprehensive cell culture studies were performed to investigate the anti-cancer effects of ABE in SW480 and HCT116 CRC cell lines. Subsequently, these findings were validated in patient-derived 3D organoids (PDOs) models. ResultsTranscriptomic profiling analysis identified p53 signaling as one of the key enriched pathways mediating the anti-cancer activity of ABE. Analysis of public datasets revealed that Chk1, a key regulator of p53, was one of the critical targets of ABE in CRC. Chk1 and p53 activation was shown to be downregulated with ABE treatment, leading to the induction of cell cycle arrest (p = 0.003–0.014) and enhanced DNA damage (p = 0.015–0.026). Furthermore, these findings were validated in PDOs, where the ABE treatment resulted in significantly fewer and smaller PDOs in a concentration-dependent manner (p = 0.045 - <0.001). ConclusionsWe firstly provide evidence for the role of the p53 signaling pathway as a mediator of the anti-cancer activity of ABE, which provides a rationale for its use as a safe and effective integrative medicine approach in CRC.

Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.

Remodelin delays non-small cell lung cancer progression by inhibiting NAT10 via the EMT pathway.

Lung cancer remains the foremost reason of cancer-related mortality, with invasion and metastasis profoundly influencing patient prognosis. N-acetyltransferase 10 (NAT10) catalyzes the exclusive N (4)-acetylcytidine (ac4C) modification in eukaryotic RNA. NAT10 dysregulation is linked to various diseases, yet its role in non-small cell lung cancer (NSCLC) invasion and metastasis remains unclear. Our study delves into the clinical significance and functional aspects of NAT10 in NSCLC. We investigated NAT10's clinical relevance using The Cancer Genome Atlas (TCGA) and a group of 98 NSCLC patients. Employing WB, qRT-PCR, and IHC analyses, we assessed NAT10 expression in NSCLC tissues, bronchial epithelial cells (BECs), NSCLC cell lines, and mouse xenografts. Further, knockdown and overexpression techniques (siRNA, shRNA, and plasmid) were employed to evaluate NAT10's effects. A series of assays were carried out, including CCK-8, colony formation, wound healing, and transwell assays, to elucidate NAT10's role in proliferation, invasion, and metastasis. Additionally, we utilized lung cancer patient-derived 3D organoids, mouse xenograft models, and Remodelin (NAT10 inhibitor) to corroborate these findings. Our investigations revealed high NAT10 expression in NSCLC tissues, cell lines and mouse xenograft models. High NAT10 level correlated with advanced T stage, lymph node metastasis and poor overall survive. NAT10 knockdown curtailed proliferation, invasion, and migration, whereas NAT10 overexpression yielded contrary effects. Furthermore, diminished NAT10 levels correlated with increased E-cadherin level whereas decreased N-cadherin and vimentin expressions, while heightened NAT10 expression displayed contrasting results. Notably, Remodelin efficiently attenuated NSCLC proliferation, invasion, and migration by inhibiting NAT10 through the epithelial-mesenchymal transition (EMT) pathway. Our data underscore NAT10 as a potential therapeutic target for NSCLC, presenting avenues for targeted intervention against lung cancer through NAT10 inhibition.

Mirk/Dyrk1B Kinase Inhibitors in Targeted Cancer Therapy.

During the last years, there has been an increased effort in the discovery of selective and potent kinase inhibitors for targeted cancer therapy. Kinase inhibitors exhibit less toxicity compared to conventional chemotherapy, and several have entered the market. Mirk/Dyrk1B kinase is a promising pharmacological target in cancer since it is overexpressed in many tumors, and its overexpression is correlated with patients' poor prognosis. Mirk/Dyrk1B acts as a negative cell cycle regulator, maintaining the survival of quiescent cancer cells and conferring their resistance to chemotherapies. Many studies have demonstrated the valuable therapeutic effect of Mirk/Dyrk1B inhibitors in cancer cell lines, mouse xenografts, and patient-derived 3D-organoids, providing a perspective for entering clinical trials. Since the majority of Mirk/Dyrk1B inhibitors target the highly conserved ATP-binding site, they exhibit off-target effects with other kinases, especially with the highly similar Dyrk1A. In this review, apart from summarizing the data establishing Dyrk1B as a therapeutic target in cancer, we highlight the most potent Mirk/Dyrk1B inhibitors recently reported. We also discuss the limitations and perspectives for the structure-based design of Mirk/Dyrk1B potent and highly selective inhibitors based on the accumulated structural data of Dyrk1A and the recent crystal structure of Dyrk1B with AZ191 inhibitor.

Abstract 7192: Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is a lethal malignancy that is partly attributable to the acquisition of chemoresistance to conventional chemotherapies. Hence, developing strategies to overcome such chemoresistance is essential. Teriflunomide, an active metabolite of leflunomide, has been widely utilized in managing autoimmune disorders, and emerging evidence suggests that it can augment the efficacy of chemotherapy in cancer. We investigated the therapeutic effect of teriflunomide in overcoming resistance to Gemcitabine-based therapy in PC. Methods: We performed docking and genomewide transcriptomic profiling analyses to identify therapeutic targets of Teriflunomide associated with Gemcitabine resistance (Gem-R) in PC. Subsequently, we analyzed the TCGA dataset to determine the prognostic significance of candidate genes in PC patients. A series of experiments were performed in PC cells, and the key findings were validated in patient-derived 3D organoids and a Gem-R Mia-PaCa2 xenograft model. Results: Docking and transcriptomic profiling studies identified GSK3β, MAPK8, and MAPK9 as potential targets of Teriflunomide, which were significantly upregulated in Gem-R cell lines (P &amp;lt; 0.05). GSK3β was independently associated with poor overall and disease-free survival in PC patients. Combined Teriflunomide and Gemcitabine treatment exhibited superior synergistic anti-tumor potential, as evidenced by reducing PC cell proliferation, clonogenicity, invasion, and migration. The combined treatment with Teriflunomide and Gemcitabine primarily inhibited GSK3β phosphorylation, resulting in the silencing of the downstream AKT/mTOR pathway. Lentiviral-based silencing of GSK3β inhibited the AKT/mTOR pathway and reduced the IC50s of Gemcitabine in PC cells. Furthermore, co-administrating both drugs to Gem-R Mia-PaCa-2 xenograft mice significantly reduced tumor growth rates and volumes. These findings were recapitulated in patient-derived 3D-organoids, where the combined treatment resulted in fewer and smaller organoids vs. each drug individually (P &amp;lt; 0.05). Conclusions: We provide novel evidence that Teriflunomide helps overcome resistance to Gemcitabine by inhibiting the GSK3β/AKT/mTOR pathway, which could improve treatment outcomes in PC. Citation Format: Caiming Xu, Yuan Li, Silei Sui, Meiling Chen, Laleh Melstorm, Haiyong Han, Steven T. Rosen, Ajay Goel. Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7192.

Abstract 6708: MAIT engagers: Bispecific antibody-mediated redirection of mucosal associated invariant T cells to treat solid tumors

Abstract Background. Using bispecific antibodies (BiXAb) to redirect T cells to engage and kill cancer cells is an efficacious modality in the treatment of haematological cancers but has had limited success in the treatment of solid tumors. Mucosal-Associated Invariant T cells (MAITs) are an abundant subset of non-conventional T-cells with potent cytotoxic capacity (up to 20% of circulating T-cells) that are naturally resident in many tissues and solid tumors. MAIT cells utilize a semi-invariant TCR and recognize bacterial metabolites presented in the context of the MR1 protein. Biomunex has generated bispecific antibodies that bind the MAIT semi-invariant TCR (iTCR) and the HER2 receptor tyrosine kinase expressed on tumor cells. At difference to classical T-cell engagers that activate all T-cell subsets, MAIT engagers only modulate the activity of the cytotoxic MAIT cells leading to efficient tumor control and greater safety profile. Methods. Using the Biomunex proprietary BiXAb platform, bispecific, tetravalent antibodies were generated that target the MAIT iTCR and the HER2. MAIT-cell activation, proliferation and degranulation were followed by gating on MAIT cells within a purified CD8 cell population. Tumor cell lines (varying in [HER2] expression) were co-cultured with MAIT cells and the BiXAbs in several cytotoxic assays (evaluated by Chromium release). Cytokine release was assessed by Legend Plex assays or ELISA. Cytotoxicity of tumor-resident MAITs, from fresh patient samples, was determined by impedence measurements and in patient-derived 3D organoids by The Hub organoids (imaging). For in vivo analysis, sub-cutaneous tumor growth was measured with callipers. Results. MAIT engagers induce the rapid activation, proliferation and degranulation of MAIT cells leading to efficient killing of engaged HER2+ cancer cells with a potency similar to that of a classical HER2-directed TCE (in clinical development). The MAIT engager did not activate the regulatory T cells or other CD4/CD8 subsets. In addition, using PBMCs, total cytokine release was 1000-fold less with the MAIT engager compared to a classical TCE, opening up a large therapeutic window, which should permit effective treatment of solid tumors. Ex vivo cytotoxicity of tumor-resident MAIT cells using dissociated ovarian cancer samples at “real E:T ratios”, MAIT engager-mediated cytotoxicity of patient-derived organoids (CRC) and in vivo evaluation of tumor control will be shown. Conclusions. MAIT cell redirection is a promising approach for the treatment of solid tumors that has the potency and efficacy of a classical TCE but with a significantly improved cytokine safety profile leading to a larger therapeutic window. Citation Format: Simon Edward Plyte, Marie Fraudeau, Dorothee Winterberg, Claire Germain, Camille Rousseau, Gaetano Sodaro, Lise Fenou, Maxime Audin, Alexandre Ivagnes, Han-Heinrich Oberg, Pierre Emmanuel Gerard, Isabelle Navarro-Teulon, Daniela Wesch, Matthias Peipp, Julie Prigent. MAIT engagers: Bispecific antibody-mediated redirection of mucosal associated invariant T cells to treat solid tumors [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 6708.

Abstract 4748: Aronia berry overcomes gemcitabine resistance by regulating the MYD88/NF-kB/P-glycoprotein pathway in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is lethal, which is partly due to the limited efficacy of Gemcitabine (Gem)-based chemotherapy, resulting from the development of acquired resistance to this regimen. Aronia berry extract (ABE) is a natural source of phenolic compounds known for its anti-cancer properties. Previous research has highlighted the ability of ABE to help overcome chemoresistance to conventional chemotherapies in various cancers. In this study, we interrogated the ability of ABE to overcome Gem-resistance (Gem-R) in PDAC and identify specific pathway(s) responsible for this anti-cancer efficacy. Methods: We performed a series of in-vitro experiments in Gem-R cells to evaluate the synergistic effect of combined treatment with Gem and ABE. Furthermore, we conducted a genomewide transcriptomic analysis to identify the critical regulatory pathways associated with chemoresistance and potential therapeutic targets of ABE in Gem-R PDAC cells. The cell culture findings were validated in patient-derived 3D organoids. Results: The combined treatment with ABE and Gem in Gem-R cells exhibited significant synergistic anticancer effects on cell viability, proliferation, migration, and invasion. The transcriptomic analysis revealed that the NF-κB signaling pathway was associated with Gem-R (p&amp;lt;0.05), with a significant upregulation of MYD88. Additionally, MYD88 was significantly associated with overall survival in PDAC patients in the TCGA cohort (HR=1.58, P=0.045). The MYD88/NF-κB signaling is associated with chemoresistance as it can upregulate the efflux transporters like P-glycoprotein (P-gp). Our results confirmed that combined treatment inhibited the NF-κB signaling pathway by suppressing MYD88 and downregulating P-gp expression to overcome Gem-R. Finally, the combined treatment demonstrated superior anti-cancer activity in 3D organoids by reducing both their number and size (P&amp;lt;0.05). Conclusion: We provide novel insights into the potential of ABE in overcoming Gem-R in PDAC cells by targeting the MYD88/NF-κb/P-gp axis, which offers a safe and inexpensive approach for improving therapeutic outcomes in this fatal malignancy. Citation Format: Yuan Li, Caiming Xu, Haiyong Han, Ajay Goel. Aronia berry overcomes gemcitabine resistance by regulating the MYD88/NF-kB/P-glycoprotein pathway in pancreatic ductal adenocarcinoma [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 4748.

Abstract 523: Aronia berry and oligomeric proanthocyanidins synergistically exhibit anticancer effects in colorectal cancer by inhibiting LMNB1

Abstract Background: Colorectal cancer (CRC) is one of the most common and recurrent diseases globally, ranking as the second leading cause of mortality. The high mortality rates associated with this disease are largely due to the ineffectiveness of current treatments and the development of chemotherapeutic resistance to conventional therapies. In recent years, several natural agents, such as Aronia berry extract (ABE) and oligomeric proanthocyanidins (OPCs), have shown promise in providing a safe, cost-effective, and synergistically multi-targeted treatment approach across various cancers. Herein, we hypothesized that the combined treatment with ABE and OPCs may synergistically regulate multiple oncogenic pathways, thereby presenting a more potent anti-cancer activity, particularly targeting CRC. Methods: We performed a series of cell culture experiments to evaluate the synergistic anticancer effect of the combination of ABE and OPCs in CRC cell lines. By performing genome-wide transcriptomic profiling, we identified specific growth-signaling pathways targeted by the combined treatment and subsequently validated these findings in cell lines and patient-derived 3D organoids. Results: The combined treatment with ABE and OPCs exhibited superior synergistic anti-tumor potential, affecting cell viability, proliferation, migration, and invasion in CRC cells. The transcriptomic analysis identified that LMNB1, associated with the cell apoptosis pathway Fold enrichment [FC]: 4.29, P =0.005), plays a crucial role in the anti-tumorigenic effects of these two natural products. Moreover, the combination of ABE and OPCs significantly altered the expression of key proteins involved in cell apoptosis and suppressed the expression level of LMNB1 in HCT116 (Combination vs. ABE, FC= 0.28; Combination vs. OPCs, FC= 0.24) and SW480 (Combination vs. ABE, FC= 0.65; Combination vs. OPCs, FC= 0.47), thereby inhibiting downstream Akt phosphorylation. Finally, we successfully demonstrated that the combined treatment displayed a superior anti-cancer activity in 3D organoids, significantly reducing both their number and size (P &amp;lt; 0.05). Conclusion: We present novel evidence for the synergistic anticancer effects of ABE and OPCs in CRC cells, which are in part achieved by regulating cellular apoptosis and the oncogene LMNB1 within the Akt signaling pathway. Citation Format: Yuan Li, Caiming Xu, Ajay Goel. Aronia berry and oligomeric proanthocyanidins synergistically exhibit anticancer effects in colorectal cancer by inhibiting LMNB1 [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 523.

Aberrant Glycosylation in Pancreatic Ductal Adenocarcinoma 3D Organoids Is Mediated by KRAS Mutations.

Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells, to gasp the comprehensive cancer cell surface glycan expression profile using lectin microarray and transcriptomic analyses. Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. We successfully established 16 3D organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS was mutated in 13 (7 G12V, 5 G12D, 1 Q61L) and wild in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Lectin reactivity of AAL (Aleuria aurantia) and AOL (Aspergillus oryzae) with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. Meanwhile, mannose-binding lectin (rRSL [Ralstonia solanacearum] and rBC2LA [Burkholderia cenocepacia]) signals were higher while those of galactose-binding lectins (rGal3C and rCGL2) were lower in the KRAS mutants. We demonstrated here that PDAC 3D-cultured organoids with KRAS mutations were dominantly covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers.

XQZ3, a Chlorella pyrenoidosa polysaccharide suppresses cancer progression by restraining mitochondrial bioenergetics via HSP90/AKT signaling pathway

The mitochondria are known to exert significant influence on various aspects of cancer cell physiology. The suppression of mitochondrial function represents a novel avenue for the advancement of anti-cancer pharmaceuticals. The heat shock protein HSP90 functions as a versatile regulator of mitochondrial metabolism in cancer cells, rendering as a promising target for anticancer interventions. In this work, a novel acid polysaccharide named as XQZ3 was extracted from Chlorella pyrenoidosa and purified by DEAE-cellulose and gel-filtration chromatography. The structural characteristic of XQZ3 was evaluated by monosaccharides composition, methylation analysis, TEM, FT-IR, and 2D-NMR. It was found that XQZ3 with a molecular weight of 29.13 kDa was a complex branched polysaccharide with a backbone mainly composed of galactose and mannose. It exhibited good antitumor activity in vitro and in vivo by patient-derived 3D organoid models and patient-derived xenografts models. The mechanistic investigations revealed that XQZ3 specifically interacted with HSP90, impeding the activation of the HSP90/AKT/mTOR signaling cascade. This, in turn, led to the induction of mitochondrial dysfunction, autophagy, and apoptosis, ultimately resulting in the demise of cancer cells due to nutrient deprivation. This study offers a comprehensive theoretical foundation for the advancement of XQZ3, a novel polysaccharide inhibitor targeting HSP90, with potential as an effective therapeutic agent against cancer.

Abstract B099: Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations

Abstract Introduction: Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. Moreover, as glycans constitute the outermost layer of cells, diverse alterations during carcinogenesis affect the multiple processes of invasion and metastasis, making glycosylation a keystone mechanism in cancer progression. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells for comprehensive cell surface glycan expression profile using lectin microarray and transcriptomic analyses and then analyzed the relationship between altered glycans and glycan-related genes, with a particular focus on the status of KRAS mutations. Materials and methods: Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system containing extracellular matrix and specific niche factors. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. Results: We successfully established 16 3D-organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS mutations (7 G12V, 5 G12D, 1 Q61L) were found in 13 organoids, while wild-type KRAS without mutations was detected in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Pairings of a PDAC organoid with a non-PDAC counterpart were available from 2 patients; one was a PDAC-normal MPD pair and the other was PDAC-IPMN pair, eliminating the influence of individual variations in glycan expression status for those pairs. Lectin reactivity of AAL and AOL with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. And also, in the two cases with PDAC and non-PDAC organoids pairs, the reactivity of AAL were significantly elevated in the KRAS mutants, with FUT6 and FUT3 elevated. Conclusion: PDAC 3D-cultured organoids with KRAS mutations were covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers. Citation Format: Hiromitsu Nakahashi, Tatsuya Oda, Osamu Shimomura, Yoshimasa Akashi, Kazuhiro Takahashi, Yoshihiro Miyazaki, Tomoaki Furuta, Yukihito Kuroda, Pakavarin Louphrasitthiphol, Bryan J. Mathis, Hiroaki Tateno. Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations [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 B099.

Innovative platforms for development of TCR-T cell therapies against novel tumor targets: Triple negative breast cancer.

e14536 Background: Adoptive T cells therapies have shown great potential over the past decade, particularly with the use of CAR-T cells to treat haematological tumors. However, their effect in the treatment of solid cancers is limited, due to a lack of relevant therapeutic targets and suppressive mechanisms of the tumor microenvironment. To counteract these challenges, we have developed Pan Cancer T (PCT) platforms to yield novel TCR-T cell therapies against untapped (intracellular) tumor targets exclusively expressed in 10 hard-to-treat solid tumors (including breast, melanoma, colorectal, glioma, lung cancer). Furthermore, we are developing engineering strategies aiming to improve the longevity of T cells within the inhibitory solid tumor microenvironment. Methods: The platforms we have developed to enable generation of genetically engineered TCR-T cells to recognize tumor-specific (intracellular) antigens include: 1) PCT Target Discovery (identifying and selecting safe, immunogenic tumor targets by combining advanced in silico and laboratory techniques); 2) PCT TCR Discovery (identifying, selecting, and validating potent TCR-T cells binding with high specificity and avidity); 3) PCT Next Gen Engineering (identifying drivers of immune evasion and implementing TCR-T cell-based gene-engineering approaches to overcome suppression and/or improve durability). Results: Using our PCT Target Discovery platform, we have validated a set of 30 unique tumor targets which are safe, immunogenic and highly shared. Our first showcase target is PCT-1, which is of particular interest in triple negative breast cancer (TNBC), where it is expressed by &gt;85% of patients, with high homogeneity in &gt;80% of all tumor cells. PCT-1 is also expressed in other tumor types, including &gt;90% melanoma and &gt;45% multiple myeloma patients. The PCT TCR Discovery platform has enabled development of a highly specific and active TCR against PCT-1, which outperformed standard of care TNBC treatments (ie, cis-platinum and sacituzumab govitecan) in advanced preclinical efficacy models, including in vitro killing of patient-derived 3D organoids and an in vivo model in NSG mice. Conclusions: The PCT platforms have enabled development of a promising TCR-T against a novel target antigen expressed in TNBC, an aggressive form of breast cancer that affects younger women with limited treatment options. A portfolio of 29 additional untapped targets has also been identified, providing potential to generate TCR-based therapies for treatment against a much larger range of solid tumors. Furthermore, we are developing next generation technologies, aiming to enhance the durability of responses within the hostile tumor microenvironment, which should be broadly applicable to TCR-T therapies.

Ginseng mediates its anticancer activity by inhibiting the expression of DNMTs and reactivating methylation-silenced genes in colorectal cancer.

Developing safe and effective therapeutic modalities remains a critical challenge for improving the prognosis of patients with colorectal cancer (CRC). In this regard, targeting epigenetic regulation in cancers has recently emerged as a promising therapeutic approach. Since several natural compounds have recently been shown to be important epigenetic modulators, we hypothesized that Ginseng might exert its anticancer activity by regulating DNA methylation alterations in CRC. In this study, a series of cell culture studies were conducted, followed by their interrogation in patient-derived 3D organoid models to evaluate Ginseng's anticancer activity in CRC. Genome-wide methylation alterations were interrogated by undertaking MethylationEpic BeadChip microarrays. First, 50% inhibitory concentrations (IC50) were determined by cell viability assays, and subsequent Ginseng treatment demonstrated a significant anticancer effect on clonogenicity and cellular migration in CRC cells. Treatment with Ginseng potentiated cellular apoptosis through regulation of apoptosis-related genes in CRC cells. Furthermore, Ginseng treatment downregulated the expression of DNA methyltransferases (DNMTs) and decreased the global DNA methylation levels in CRC cells. The genome-wide methylation profiling identified Ginseng-induced hypomethylation of transcriptionally silenced tumor suppressor genes. Finally, cell culture-based findings were successfully validated in patient-derived 3D organoids. In conclusion, we demonstrate that Ginseng exerts its antitumorigenic potential by regulating cellular apoptosis via the downregulation of DNMTs and reversing the methylation status of transcriptionally silenced genes in CRC.

Abstract 3821: Berberine and emodin synergistically suppress the EGFR signaling cascade by targeting LAMB3 in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic cancer is one of the most devastating malignancies due to the development of intrinsic chemoresistance following chemotherapy. Extracellular matrix (ECM) proteins are intimately linked to cellular proliferation, invasion, and acquisition of chemoresistance in PDAC cells, making them promising therapeutic targets in this malignancy. Naturally occurring dietary botanicals, including berberine (BER) and emodin (EMO), have been shown to suppress ECM as one of the mechanism(s) for their anti-tumorigenic activity, along with their time-tested safety and cost-effectiveness. In addition, both BER and EMO are also known to induce apoptosis by modulating different pathways and regulating pro-apoptotic genes. Herein, we hypothesized that combined treatment with BER and EMO might exhibit synergistic anticancer efficacy by targeting the ECM and apoptotic pathways in PDAC cells. Methods: We undertook genomewide transcriptomic profiling analysis to identify critical ECM-related genes differentially expressed in PDAC. Subsequently, the TCGA dataset was analyzed to identify the prognostic significance of ECM-associated genes with overall survival (OS) and disease-free survival (DFS) in PDAC. A series of cell culture experiments were performed using PDAC cells, followed by their validation in patient-derived organoids to examine the synergistic anti-proliferative and chemopreventive effects of BER and EMO against PDAC. Results: Transcriptomic profiling identified that LAMB3 expression was significantly upregulated in PDAC tissue (P &amp;lt; 0.01) and was significantly associated with poor OS and DFS in PDAC patients (P &amp;lt; 0.01). The combination of BER and EMO displayed superior synergistic anti-tumor potential in PDAC cells vs. individual compounds, as revealed by cell proliferation, clonogenicity, migration, and invasion assays. The combination of BER and EMO also altered the expression of key proteins involved in cellular apoptosis, epithelial-mesenchymal-transition, and EGFR/ERK//AKT growth factor signaling pathways. Finally, these findings were successfully validated in PDAC patient-derived 3D organoids. Conclusions: We provide the first evidence that combined treatment with berberine and emodin exerts synergistic anti-cancer activity in PDAC, primarily regulated through the LAMB3-mediated interaction with other members of the EGFR-signaling pathway. Citation Format: CAIMING XU, SILEI SUI, Keisuke Okuno, Silvia Pascual-Sabater, Cristina Fillat, Ajay Goel. Berberine and emodin synergistically suppress the EGFR signaling cascade by targeting LAMB3 in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3821.

Use of a pulmosphere model to evaluate drug antifibrotic responses in interstitial lung diseases

BackgroundInterstitial lung diseases (ILD) encompass a heterogenous group of diffuse parenchymal lung disorders characterized by variable degrees of inflammation and fibrosis. Pretherapeutic clinical testing models for such diseases can serve as a platform to test and develop effective therapeutic strategies. In this study, we developed patient derived 3D organoid model to recapitulate the disease process of ILDs. We characterized the inherent property of invasiveness in this model and tested for antifibrotic responses with an aim to develop a potential platform for personalized medicine in ILDs.MethodsIn this prospective study, 23 patients with ILD were recruited and underwent lung biopsy. 3D organoid-based models (pulmospheres) were developed from the lung biopsy tissues. Pulmonary functioning testing and other relevant clinical parameters were collected at the time of enrollment and follow up visits. The patient derived pulmospheres were compared to normal control pulmospheres obtained from 9 explant lung donor samples. These pulmospheres were characterized by their invasive capabilities and responsiveness to the antifibrotic drugs, pirfenidone and nintedanib.ResultsInvasiveness of the pulmospheres was measured by the zone of invasiveness percentage (ZOI%). The ILD pulmospheres (n = 23) had a higher ZOI% as compared to control pulmospheres (n = 9) (516.2 ± 115.6 versus 54.63 ± 19.6 respectively. ILD pulmospheres were responsive to pirfenidone in 12 of the 23 patients (52%) and responsive to nintedanib in all 23 patients (100%). Pirfenidone was noted to be selectively responsive in patients with connective tissue disease related ILD (CTD-ILD) at low doses. There was no correlation between the basal pulmosphere invasiveness, response to antifibrotics, and FVC change (Δ FVC).ConclusionsThe 3D pulmosphere model demonstrates invasiveness which is unique to each individual subject and is greater in ILD pulmospheres as compared to controls. This property can be utilized to test responses to drugs such as antifibrotics. The 3D pulmosphere model could serve as a platform for the development of personalized approaches to therapeutics and drug development in ILDs and potentially other chronic lung diseases.

Andrographis Reverses Gemcitabine Resistance through Regulation of ERBB3 and Calcium Signaling Pathway in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, primarily due to intrinsic or acquired resistance to chemotherapy, such as Gemcitabine (Gem). Naturally occurring botanicals, including Andrographis (Andro), can help enhance the anti-tumorigenic therapeutic efficacy of conventional chemotherapy through time-tested safety and cost-effectiveness. Accordingly, we hypothesized that Andro might reverse Gem resistance in PDAC. The critical regulatory pathways associated with Gem resistance in PDAC were identified by analyzing publicly available transcriptomic profiling and PDAC tissue specimens. A series of systematic in vitro experiments were performed using Gem-resistant (Gem-R) PDAC cells and patient-derived 3D-organoids to evaluate the Andro-mediated reversal of Gem resistance in PDAC. Transcriptomic profiling identified the calcium signaling pathway as a critical regulator of Gem-resistance (Fold enrichment: 2.8, p = 0.002). Within this pathway, high ERBB3 expression was significantly associated with poor prognosis in PDAC patients. The combination of Andro and Gem exhibited superior anti-cancer potential in Gem-R PDAC cells through potentiating cellular apoptosis. The combined treatment down-regulated ERBB3 and decreased intracellular calcium concentration in Gem-R PDAC cells. Finally, these findings were successfully interrogated in patient-derived 3D-organoids. In conclusion, we demonstrate novel evidence for Andro-mediated reversal of chemoresistance to Gem in PDAC cells through the regulation of ERBB3 and calcium signaling.

Establishment of a direct 2.5D organoid culture model using companion animal cancer tissues

Like humans, cancer affects companion animals with similar genetic risks and incident rates. To improve treatment strategies for pet cancers, new research models are necessary. Patient-derived 3D organoid culture models are valuable and ensure the development of new effective therapies. In the previous study, we established a 3D organoid-derived 2.5D organoid culture model that recapitulated some characteristics of their parental 3D organoids. In the present study, we aimed to generate a 2.5D organoid culture model directly from cancer-diseased dogs and cats using special 2.5D media. The primary cultured cells in 2.5D media (direct 2.5D organoids) showed better attachment, growth, marker expression, and faster proliferation speed than those cultured in normal Dulbecco’s Modified Eagle Medium media. The direct 2.5D organoids showed expression of each specific marker to their original cancer tissues and exhibited tumorigenesis in vivo. Moreover, the direct 2.5D organoids exhibited concentration-dependent responses to anti-cancer drugs, and different sensitivity profiles were shown among the strains. Our data suggest that the direct 2.5D organoid culture model might become a useful tool beyond 2D cell lines to study cancer biology in companion animals and could provide new platforms for screening the anti-cancer drugs.

Microfluidics Formulated Liposomes of Hypoxia Activated Prodrug for Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) presents as an unmet clinical challenge for drug delivery due to its unique hypoxic biology. Vinblastine-N-Oxide (CPD100) is a hypoxia-activated prodrug (HAP) that selectively converts to its parent compound, vinblastine, a potent cytotoxic agent, under oxygen gradient. The study evaluates the efficacy of microfluidics formulated liposomal CPD100 (CPD100Li) in PDAC. CPD100Li were formulated with a size of 95 nm and a polydispersity index of 0.2. CPD100Li was stable for a period of 18 months when freeze-dried at a concentration of 3.55 mg/mL. CPD100 and CPD100Li confirmed selective activation at low oxygen levels in pancreatic cancer cell lines. Moreover, in 3D spheroids, CPD100Li displayed higher penetration and disruption compared to CPD100. In patient-derived 3D organoids, CPD100Li exhibited higher cell inhibition in the organoids that displayed higher expression of hypoxia-inducible factor 1 alpha (HIF1A) compared to CPD100. In the orthotopic model, the combination of CPD100Li with gemcitabine (GEM) (standard of care for PDAC) showed higher efficacy than CPD100Li alone for a period of 90 days. In summary, the evaluation of CPD100Li in multiple cellular models provides a strong foundation for its clinical application in PDAC.

Abstract PR-010: Collateral amplification of the KRAS linked gene PTHLH governs pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability

Abstract Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial, and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT both in vivo and in vitro, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Anna M. Chiarella, Kensuke Suzuki, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification of the KRAS linked gene PTHLH governs pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-010.

FSTL1 Secreted by Activated Fibroblasts Promotes Hepatocellular Carcinoma Metastasis and Stemness.

The tumor microenvironment plays a critical role in maintaining the immature phenotype of tumor-initiating cells (TIC) to promote cancer. Hepatocellular carcinoma (HCC) is a unique disease in that it develops in the setting of fibrosis and cirrhosis. This pathologic state commonly shows an enrichment of stromal myofibroblasts, which constitute the bulk of the tumor microenvironment and contribute to disease progression. Follistatin-like 1 (FSTL1) has been widely reported as a proinflammatory mediator in different fibrosis-related and inflammatory diseases. Here we show FSTL1 expression to be closely correlated with activated fibroblasts and to be elevated in regenerative, fibrotic, and disease liver states in various mouse models. Consistently, FSTL1 lineage cells gave rise to myofibroblasts in a CCL4-induced hepatic fibrosis mouse model. Clinically, high FSTL1 in fibroblast activation protein-positive (FAP+) fibroblasts were significantly correlated with more advanced tumors in patients with HCC. Although FSTL1 was expressed in primary fibroblasts derived from patients with HCC, it was barely detectable in HCC cell lines. Functional investigations revealed that treatment of HCC cells and patient-derived 3D organoids with recombinant FSTL1 or with conditioned medium collected from hepatic stellate cells or from cells overexpressing FSTL1 could promote HCC growth and metastasis. FSTL1 bound to TLR4 receptor, resulting in activation of AKT/mTOR/4EBP1 signaling. In a preclinical mouse model, blockade of FSTL1 mitigated HCC malignancy and metastasis, sensitized HCC tumors to sorafenib, prolonged survival, and eradicated the TIC subset. Collectively, these data suggest that FSTL1 may serve as an important novel diagnostic/prognostic biomarker and therapeutic target in HCC. SIGNIFICANCE: This study shows that FSTL1 secreted by activated fibroblasts in the liver microenvironment augments hepatocellular carcinoma malignancy, providing a potential new strategy to improve treatment of this aggressive disease.