Patient-derived Colorectal Cancer Organoids Research Articles

EP2/EP4 targeting prevents tumor-derived PGE2-mediated immunosuppression in cDC2s.

Tumor-derived prostaglandin E2 (PGE2) impairs anti-tumor immunity by priming suppressive functions on various immune cell types, including dendritic cells (DCs). In this way, tumors mediate DC dysfunction and hamper their anti-tumoral activity. PGE2 is known to modulate DC function via signaling through the E-prostanoid receptor type (EP) 2 and EP4. Preclinical studies have demonstrated the therapeutic value of targeting EP2/4 receptor signaling in DCs. Ongoing phase I clinical trials with EP antagonists have shown immunomodulation in cancer patients. However, the systemic drug administration leads to off-target events and subsequent side-effects. To limit the off-target effects of EP targeting, EP2 and EP4 antagonists were encapsulated in polymeric nanoparticles (NPs). In this study we evaluated the efficacy of EP2/4 specific antagonists encapsulated in NPs to protect cDC2s from suppressive effects of tumor-derived PGE2 in different tumor models. We show that tumor-derived PGE2 signals via EP2/4 to mediate the acquisition of a suppressive phenotype of cDC2s. EP2/4 antagonists encapsulated NPs impaired the conversion of cDC2s towards a suppressive state and inhibited the occurrence of suppressive features such as IL-10 production or the ability to expand Tregs. Importantly, the NPs abolished the transition towards this suppressive state in different tumor models: Melanoma-conditioned media, ascites fluid derived from ovarian cancer patients (2D), and upon coculture with colorectal cancer patient-derived organoids (3D). We propose that targeting the PGE2-EP2/4 axis using NPs can achieve immunomodulation in the immune system of cancer patients, alleviate tumor-derived suppression, and thus facilitate the development of potent anti-tumor immunity in cancer patients.

Efficacy of fadraciclib (CYC065), a novel dual CDK2/9 inhibitor, on patient-derived models of colorectal cancer.

3596 Background: Colorectal cancer (CRC) is the third most common type of cancer among adults in the United States. Given the heterogeneous nature of the disease and the limited number of treatments available, there is an unmet need to identify new therapeutic vulnerabilities for advanced CRC. One promising approach is targeting of cyclin dependent kinases (CDKs). In our current study, we sought to understand the potential efficacy of fadraciclib (CYC065), a novel dual CDK 2/9 inhibitor, as a novel treatment for metastatic CRC. Methods: Eighteen CRC patient-derived organoids (PDOs) were generated from patients undergoing biopsy or resection for their primary or metastatic CRC under an IRB approved protocol at Duke University. IHC and H&E staining were first performed on the established PDOs to confirm the diagnosis of CRC. PDOs were treated with standard of care chemotherapy (oxaliplatin, irinotecan (SN38) and 5-Flurouracil), CDK4/6 inhibitor palbociclib, and fadraciclib (CYC065). All drugs were used in doses ranging from 6.4nM to 100µM, and drug sensitivities were determined using Cell-Titer Glo (CTG) 72 hours after treatment. Subsequently, three matching PDXs were generated for in vivo validation and treated with fadraciclib (CYC065) via oral gavage at a dose of 25 mg/kg BID five days a week for two weeks. Target validation for CDK2/9 inhibition was performed via western blotting, cell cycle arrest was determined via flow cytometry, and killing by anaphase catastrophe was determined by immunofluorescence staining. Results: CRC PDOs were more sensitive to fadraciclib (CYC065) than chemotherapy and palbociclib, with IC50 values lower than chemotherapy or palbociclib (2.65±3.92µM for fadraciclib (CYC065) vs. 29.92±41.98µM for chemotherapy (P<0.05) or palbociclib (12.33±9.87µM)). Subsequently, we validated our findings in vivo using 3 matched PDX showing significant tumor growth inhibition (TGI) with fadraciclib (CYC065) compared to control (p<0.05) without serious adverse effects. We showed that fadraciclib (CYC065) induces G2/M cell cycle arrest, resulting in anaphase catastrophe as evident by the presence of multipolar mitosis in fadraciclib (CYC065)-treated cells. CDK2/9 inhibition leads to downregulation of c-Myc levels and upregulation of apoptosis induction as a second mechanism of action for this drug. Conclusions: Our data using both in vitro and in vivo patient-derived models support further exploration of fadraciclib (CYC065) has a potential therapy for advanced CRC. CDK2/9 inhibition impacts multiple critical pathways involved in transcription, mitosis and apoptosis. Further studies will be needed to reveal molecular biomarkers of sensitivity and resistance to this novel agent.

Hyaluronic acid-conjugated methotrexate and 5-fluorouracil for targeted drug delivery

The delivery of chemotherapeutical drugs via nanomaterials has become a focus of pharmaceutical research over several decades due to improved drug delivery to cancer cells, decreased side effects on normal tissues, and increased therapeutic efficacy. Herein, a novel hyaluronic acid-conjugated methotrexate and 5-fluorouracil nanodrug system has been developed to address the critical limitations associated with the high toxicity and side effects of methotrexate and 5-fluorouracil. Furthermore, this nanodrug system enhances the targeting capacity of drug molecules and facilitates the potential integration of multimodal drug therapies. Concomitantly, the synergistic effects of MTX with 5-fluorouracil have been shown to improve the therapeutic index of MTX while attenuating the associated toxicities of MTX. The structure and micromorphology of the novel nanodrug can be confirmed by 1HNMR, FT-IR, UV–Vis, DLS, TEM, and AFM. Due to the ability of HA to bind to CD44 receptors activated on the surface of cancer cells and its enhanced permeability and retention (EPR) effect, the novel nanodrug we designed and synthesized can effectively target cancer cells. Cell counting Kit-8 (CCK8), flow cytometry, and live-dead staining assays in vitro showed that this nanodrug system had high targeting and antitumor activity against CD44 receptors. By using drugs to act on patient-derived colorectal, liver, and breast cancer organoids, the anticancer effect of the nanodrug was identified and verified. These results showed that the nanodrug system developed in this study may have great potential as a targeted therapy for cancer.

SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis

BackgroundAberrant alternative splicing (AS) is a pervasive event during colorectal cancer (CRC) development. SF3B3 is a splicing factor component of U2 small nuclear ribonucleoproteins which are crucial for early stages of spliceosome assembly. The role of SF3B3 in CRC remains unknown.MethodsSF3B3 expression in human CRCs was analyzed using publicly available CRC datasets, immunohistochemistry, qRT-PCR, and western blot. RNA-seq, RNA immunoprecipitation, and lipidomics were performed in SF3B3 knockdown or overexpressing CRC cell lines. CRC cell xenografts, patient-derived xenografts, patient-derived organoids, and orthotopic metastasis mouse models were utilized to determine the in vivo role of SF3B3 in CRC progression and metastasis.ResultsSF3B3 was upregulated in CRC samples and associated with poor survival. Inhibition of SF3B3 by RNA silencing suppressed the proliferation and metastasis of CRC cells in vitro and in vivo, characterized by mitochondria injury, increased reactive oxygen species (ROS), and apoptosis. Mechanistically, silencing of SF3B3 increased mTOR exon-skipped splicing, leading to the suppression of lipogenesis via mTOR-SREBF1-FASN signaling. The combination of SF3B3 shRNAs and mTOR inhibitors showed synergistic antitumor activity in patient-derived CRC organoids and xenografts. Importantly, we identified SF3B3 as a critical regulator of mTOR splicing and autophagy in multiple cancers.ConclusionsOur findings revealed that SF3B3 promoted CRC progression and metastasis by regulating mTOR alternative splicing and SREBF1-FASN-mediated lipogenesis, providing strong evidence to support SF3B3 as a druggable target for CRC therapy.

Abstract LB219: DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays

Abstract Gastrointestinal cancers, including gastric cancer (GC) and colorectal cancer (CRC), continue to be devastating diseases despite advances in screening, early detection, and removal of pre-neoplastic and early cancerous lesions. Even with advancements in targeted chemotherapy and immunotherapies, advanced and metastatic diseases continue to be responsible for the high mortality associated with GC and CRC. Newer, safer, and more effective therapies to prevent, inhibit, or reverse advanced disease progression are urgently needed. Doublecortin-like kinase 1 (DCLK1) is a microtubule associated protein kinase that has been reported to drive tumor initiation, progression, metastasis, and drug resistance in many solid tumor cancers including GC and CRC, where it is upregulated in patient tumors and associated with poor overall survival. An anti-DCLK1 monoclonal antibody has demonstrated potential therapeutic activity against CRC. In this study, we established patient-derived organoids (PDOs) to evaluate the effects of inhibiting DCLK1 kinase activity (DCLK1-IN-1) or targeting DCLK1 with a highly specific CAR-T cells on the treated PDOs. Two GC PDOs were treated with various concentrations of DCLK1 kinase inhibitor DCLK1-IN-1 (0-32 μM) for 72 hrs, viability of the PDOs were determined, histochemistry and immunohistochemistry staining were used to image the live cells and DCLK1 expression. For CRC PDOs, five CRC PDOs were cultured in the presence or absence of PBMC, treated with Mock CAR-T cells or DCLK1 CAR-T cells (three doses) for 4 days. Tumor size, tumor total area, and viability of these PDOs were determined. Here, we demonstrate that treating GC PDOs with DCLK1 kinase inhibitor resulted in a significant dose-dependent inhibition in PDO numbers in both PDOs (two-way ANOVA P<0.0001), a decrease of cell viability, revealing IC50 values of 7.566 and 9.958 μM respectively, and a near total loss of GC PDO viability at 10 μM using CellTiterGlo assay. In CRC PDOs, two of five CRC PDOs showed a significant decrease in total tumor size, tumor areas, and an increase in cell death measured by the DRAQ7 method. Compared to Mock CAR-T cells, treatment with DCLK1 CAR-T cells resulted in a dose dependent decrease of tumor size and total tumor area, with more than a 50% reduction at the 50000 cells dose (p<0.05). Treatment with DCLK1 CAR-T cells also resulted in a dose dependent increase in cell death, with approximately 20% cell death without PBMC, and 50% cell death with PBMC at the 50000 cells dose (p<0.05). These results suggest that targeting DCLK1 represents a potential novel therapeutic strategy for GI cancers, especially in combination with FDA approved drugs: 5-fluorouracil (5-FU) or Gefitinib. Citation Format: Dongfeng Qu, Jinsen Shi, Zhiyun Cao, Eddie BAnnerman-Menson, Nathaniel Weygant, Courtney Houchen. DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB219.

Anticolorectal Cancer Activity of Bilobalide in Patient-Derived Colorectal Cancer Organoids and AOM/DSS Mouse Model

Bilobalide has shown strong anti-inflammatory activity. Colorectal cancer (CRC) is closely associated with inflammation. However, no studies have reported on the use of bilobalide for treating CRC. This study aims to evaluate the effect of bilobalide on CRC prevention. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (RT-qPCR), Western blotting, and immunofluorescence showed that bilobalide significantly inhibits the M2 polarization of macrophages dependent on phorbol 12-myristate 13-acetate (PMA) and interleukin-4 (IL-4). Analysis of signaling pathways showed that the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT3) was regulated. In particular, human CRC organoids were established. Western blotting, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and analysis of cell viability and morphology further supported the hypothesis that the anti-CRC effects of bilobalide could be explained by its ability to suppress M2 macrophage polarization and promote M1 transformation. C57BL/6 mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) were divided into three groups, i.e., control, AOM/DSS, low (2.5 mg/kg), and high (5 mg/kg). High-dose bilobalide markedly inhibited the progression of CRC, as evidenced by the increased body weight, decrease in disease activity index (DAI) death rate, and alleviation of colon length reduction and tumorigenesis. According to the in vivo results, reduced levels of inflammatory cytokines in the serum included tumor necrosis factor (TNF-α), IL-6, IL-1β, and IL-10. Bilobalide reduced oxidative stress indices, lipid peroxide (LPO), and malondialdehyde (MDA) and increased reduced glutathione (GSH). In addition, the expression of proliferating cell nuclear antigen (PCNA), Ki67, cellular Myc (c-Myc), and CD206 was downregulated in the drug-treated groups, as confirmed by the immunohistochemical staining. Collectively, these results indicated that bilobalide administration improve experimental CRC by inhibiting M2 macrophage polarization and oxidative stress. Thus, bilobalide may prevent CRC and serve as a potential therapeutic target for CRC.

Abstract 6724: Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture model

Abstract In the field of cancer immunotherapy, bispecific antibodies have emerged as a promising approach for redirecting T-cells to target and combat tumors. However, their efficacy is limited to only a subset of patients. A major challenge that hinders the development of bispecific antibody targets is the lack of preclinical models that preserve patient-specific tumor antigens and recapitulate the complex interaction between the human immune system and tumor cells. HUB Organoids technology presents a valuable platform for novel immunotherapies testing and validation as they are directly derived from patient tumor tissues, accurately reflecting patient-specific tumor antigens. BIOMUNEX Pharmaceuticals has developed the BiXAb® technology platform, a computational modeling approach that rapidly generates bispecific antibodies from various monospecific monoclonal antibodies, eliminating the need for extensive engineering. This platform includes a bivalent T cell engager specifically targeting MAIT cells and human epithelial growth factor receptor (HER2), a tumor antigen highly expressed in some colorectal cancer (CRC) patients. This innovative approach forms an efficient immunological synapse, allowing exclusive redirection of MAIT cells to directly eliminate cancer cells. In a collaborative research initiative between HUB Organoids® and BIOMUNEX Pharmaceuticals, the mechanisms by which BiXAb® induce tumor cell killing were confirmed using CRC patient-derived organoids (HUB Organoids® or PDOs) co-cultured with allogenic MAIT cells in an in vitro model. The study results depicted BiXAb® induced MAIT-cell activation in a dose-dependent manner, as detected by FACS and ELISA. Furthermore, BiXAb®-mediated MAIT-cell-specific killing of CRC PDOs was observed in a HER2-dependent manner, determined using an image-based viability assay. In conclusion, the co-culture platform of CRC PDOs-MAIT cells described here effectively captured the activation and targeted killing of MAIT cells mediated by BiXAb®, underscoring the added value of this PDO-based co-culture platform in developing novel immunotherapies. Citation Format: Yasmine Abouleila, Julie Prigent, Mayke Doorn, Claire Germain, Sylvia Boj, Simon Plyte, Carla Verissimo. Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture 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 6724.

Abstract 1514: A novel workflow to assess the T-cell and patient-derived organoid interaction

Abstract Immunotherapy is increasingly popular as a type of cancer treatment. These therapies include the use of Chimeric Antigen Receptor-engineered T-cells (CAR T-cells), tumor-infiltrating lymphocytes (TIL), and other genetically modified T-cells to specifically target the cancer cells. Although much success has been achieved with immunotherapy for treatment of blood cancers, its efficacy remains limited in solid tumors. One of the reasons for the low success rate is attributed to the solid tumor microenvironment (TME) where suppressive cytokines limit the tumor killing ability of T-cells. Thus, understanding the role the TME plays in T-cell responses is essential for the development of effective cancer therapies. The benefits of using 3-dimensional (3D) PDOs lie in the physical and chemical cues present within the TME that cannot be mimicked in traditional 2D monolayer cultures. Studies show that PDOs show similar responses to drugs as original tumors, suggesting the value of using PDOs to improve therapeutic outcomes. Thus, PDOs can provide more relevant physiological and pathological cancer models that recapitulate the basic features of primary tumors and is more suited for assessing the effectiveness of T-cell killing than 2D cell models. Despite the benefits associated with the use of PDOs, there are significant barriers to widespread adoption of PDOs in drug discovery. Organoid production is a costly and highly labor-intensive process. Moreover, organoid culture is a skilled manual process, and thus there can be significant variability between operators. To address the challenges associated with the use of PDOs in large scale applications, a semi-automated bioprocess has been developed for the large-scale expansion of assay ready organoids. Here, we developed a method to assess the effectiveness of T-cell invasion in solid tumors using patient derive organoids (PDOs). Using bioreactor expanded patient-derived colorectal cancer organoids (CRCs), activated PBMCs (human peripheral blood mononuclear cells) stained with CellTracker were added to CRCs (stained with MitoTracker) in a 96well microtiter plate and monitored on every 4 hours for 3 days using high content imager. To quantify T-cell invasion, we developed an image analysis method to measure the distance of each T-cell to the nearest organoid (interaction distance). We find that stimulated T-cell resulted in smaller interaction distance than non-stimulated T-cells. The results demonstrate the utility of the bioreactor-expanded organoids in large scale T-cell based screens. Citation Format: Zhisong Tong, Angeline Lim, Oksana Sirenko. A novel workflow to assess the T-cell and patient-derived organoid interaction [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 1514.

Abstract 1507: Involvement of GABAergic signaling in enhanced tumor cell invasion in a mechanically dynamic tumor microenvironment

Abstract Metastasis is a major contributor to cancer morbidity and mortality. However, studying this complex, multi-step, multi-organ process is challenging. There is a pressing need to dissect the contributions of the tumor microenvironment (TME) on this process in a controlled and precise manner. Fortunately, the utilization of microfluidic “organs-on-chips” (OOC) in cancer research is facilitating such investigations. Moreover, by combining organoids and organs-on-chips we can enhance the progress of these studies in a patient specific manner. In this study, we have developed a cancer-on-chip model aimed at investigating early metastatic spread within the colon milieu. To achieve this, we employ colorectal cancer (CRC) cell lines as well as patient-derived CRC organoids chosen from our biorepository to encompass diverse representations across race/ethnicity, sex, and mutational profiles. The tumor cells are introduced into the upper chamber of the OOC model, while human microvascular endothelial cells (HIMECs) are introduced into the lower chamber to establish a tube-like structure resembling a blood vessel. These two chambers are separated by a porous membrane, and the chip is flanked by vacuum chambers to introduce stretch-like motions, simulating peristalsis in the gastrointestinal track. Creating a mechanically dynamic microenvironment facilitates the exploration of neurotransmitters and their impact on tumor cell behavior. Recent studies have suggested that tumor cells may exploit physiological processes, such as neurotransmitter signaling, to their benefit. By utilizing mass spectrometry-based metabolomics we detected dynamic and patient organoid-specific changes in neurotransmitter levels (i.e., serotonin, aspartate, glutamate, γ-aminobutyric acid (GABA)) in the effluent of our CRC-on-chip model. Coupled with live-cell imaging, we discovered tumor cell-derived GABA, a major inhibitory neurotransmitter, serves as an energy source for supporting tumor cell intravasation. This finding was most pronounced in KRAS-mutant tumor cells and further supported by analysis of CRC patient samples from The Cancer Genome Atlas (TCGA) database. We were able to reverse the GABA-mediated invasion effect by inhibiting 4-aminobutyrate aminotransferase (ABAT), the enzyme responsible for GABA catabolism. In summary, our cancer-on-chip model holds promise for exploring various aspects of the metastatic process and uncovering potential therapeutic targets, such as neurotransmitters. Citation Format: Carly Strelez, Rachel Perez, John S. Chlystek, Christopher Cherry, Bethany Haliday, Ah Young Yoon, Curran Shah, Ren X. Sun, Roy Lau, Aaron Schatz, Josh Neman, Heinz Josef-Lenz, Jonathan Katz, Shannon M. Mumenthaler. Involvement of GABAergic signaling in enhanced tumor cell invasion in a mechanically dynamic tumor microenvironment [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 1507.

CDC2 plasticity and acquisition of a DC3-like phenotype mediated by IL-6 and PGE2 in a patient-derived colorectal cancer organoids model.

Metastatic colorectal cancer (CRC) is highly resistant to therapy and prone to recur. The tumor-induced local and systemic immunosuppression allows cancer cells to evade immunosurveillance, facilitating their proliferation and dissemination. Dendritic cells (DCs) are required for the detection, processing, and presentation of tumor antigens, and subsequently for the activation of antigen-specific T cells to orchestrate an effective antitumor response. Notably, successful tumors have evolved mechanisms to disrupt and impair DC functions, underlining the key role of tumor-induced DC dysfunction in promoting tumor growth, metastasis initiation, and treatment resistance. Conventional DC type 2 (cDC2) are highly prevalent in tumors and have been shown to present high phenotypic and functional plasticity in response to tumor-released environmental cues. This plasticity reverberates on both the development of antitumor responses and on the efficacy of immunotherapies in cancer patients. Uncovering the processes, mechanisms, and mediators by which CRC shapes and disrupts cDC2 functions is crucial to restoring their full antitumor potential. In this study, we use our recently developed 3D DC-tumor co-culture system to investigate how patient-derived primary and metastatic CRC organoids modulate cDC2 phenotype and function. We first demonstrate that our collagen-based system displays extensive interaction between cDC2 and tumor organoids. Interestingly, we show that tumor-corrupted cDC2 shift toward a CD14+ population with defective expression of maturation markers, an intermediate phenotype positioned between cDC2 and monocytes, and impaired T-cell activating abilities. This phenotype aligns with the newly defined DC3 (CD14+ CD1c+ CD163+) subset. Remarkably, a comparable population was found to be present in tumor lesions and enriched in the peripheral blood of metastatic CRC patients. Moreover, using EP2 and EP4 receptor antagonists and an anti-IL-6 neutralizing antibody, we determined that the observed phenotype shift is partially mediated by PGE2 and IL-6. Importantly, our system holds promise as a platform for testing therapies aimed at preventing or mitigating tumor-induced DC dysfunction. Overall, our study offers novel and relevant insights into cDC2 (dys)function in CRC that hold relevance for the design of therapeutic approaches.

CircHAS2 activates CCNE2 to promote cell proliferation and sensitizes the response of colorectal cancer to anlotinib

BackgroundTyrosine kinase inhibitors (TKIs) are crucial in the targeted treatment of advanced colorectal cancer (CRC). Anlotinib, a multi-target TKI, has previously been demonstrated to offer therapeutic benefits in previous studies. Circular RNAs (circRNAs) have been implicated in CRC progression and their unique structural stability serves as promising biomarkers. The detailed molecular mechanisms and specific biomarkers related to circRNAs in the era of targeted therapies, however, remain obscure.MethodsThe whole transcriptome RNA sequencing and function experiments were conducted to identify candidate anlotinib-regulated circRNAs, whose mechanism was confirmed by molecular biology experiments. CircHAS2 was profiled in a library of patient-derived CRC organoids (n = 22) and patient-derived CRC tumors in mice. Furthermore, a prospective phase II clinical study of 14 advanced CRC patients with anlotinib-based therapy was commenced to verify drug sensitivity (ClinicalTrials.gov identifier: NCT05262335).ResultsAnlotinib inhibits tumor growth in vitro and in vivo by downregulating circHAS2. CircHAS2 modulates CCNE2 activation by acting as a sponge for miR-1244, and binding to USP10 to facilitate p53 nuclear export as well as degradation. In parallel, circHAS2 serves as a potent biomarker predictive of anlotinib sensitivity, both in patient-derived organoids and xenograft models. Moreover, the efficacy of anlotinib inclusion into the treatment regimen yields meaningful clinical responses in patients with high levels of circHAS2. Our findings offer a promising targeted strategy for approximately 52.9% of advanced CRC patients who have high circHAS2 levels.ConclusionsCircHAS2 promotes cell proliferation via the miR-1244/CCNE2 and USP10/p53/CCNE2 bidirectional axes. Patient-derived organoids and xenograft models are employed to validate the sensitivity to anlotinib. Furthermore, our preliminary Phase II clinical study, involving advanced CRC patients treated with anlotinib, confirmed circHAS2 as a potential sensitivity marker.

Discovery of a nitroaromatic nannocystin with potent in vivo anticancer activity against colorectal cancer by targeting AKT1.

The development of targeted chemotherapeutic agents against colorectal cancer (CRC), one of the most common cancers with a high mortality rate, is in a constant need. Nannocystins are a family of myxobacterial secondary metabolites featuring a 21-membered depsipeptide ring. The in vitro anti-CRC activity of natural and synthetic nannocystins was well documented, but little is known about their in vivo efficacy and if positive, the underlying mechanism of action. In this study we synthesized a nitroaromatic nannocystin through improved preparation of a key fragment, and characterized its in vitro activity and in vivo efficacy against CRC. We first described the total synthesis of compounds 2-4 featuring Heck macrocyclization to forge their 21-membered macrocycle. In a panel of 7 cancer cell lines from different tissues, compound 4 inhibited the cell viability with IC values of 1-6 nM. In particular, compound 4 (1, 2, 4 nM) inhibited the proliferation of CRC cell lines (HCT8, HCT116 and LoVo) in both concentration and time dependent manners. Furthermore, compound 4 concentration-dependently inhibited the colony formation and migration of CRC cell lines. Moreover, compound 4 induced cell cycle arrest at sub-G1 phase, apoptosis and cellular senescence in CRC cell lines. In three patient-derived CRC organoids, compound 4 inhibited the PDO with IC values of 3.68, 28.93 and 11.81 nM, respectively. In a patient-derived xenograft mouse model, injection of compound 4 (4, 8 mg/kg, i.p.) every other day for 12 times dose-dependently inhibited the tumor growth without significant change in body weight. We conducted RNA-sequencing, molecular docking and cellular thermal shift assay to elucidate the anti-CRC mechanisms of compound 4, and revealed that it exerted its anti-CRC effect at least in part by targeting AKT1.

Staufen1 Represses the FOXA1-Regulated Transcriptome by Destabilizing FOXA1 mRNA in Colorectal Cancer Cells.

Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.

EP2 and EP4 blockade prevents tumor-induced suppressive features in human monocytic myeloid-derived suppressor cells.

Tumors educate their environment to prime the occurrence of suppressive cell subsets, which enable tumor evasion and favors tumor progression. Among these, there are the myeloid-derived suppressor cells (MDSCs), their presence being associated with the poor clinical outcome of cancer patients. Tumor-derived prostaglandin E2 (PGE2) is known to mediate MDSC differentiation and the acquisition of pro-tumor features. In myeloid cells, PGE2 signaling is mediated via E-prostanoid receptor type 2 (EP2) and EP4. Although the suppressive role of PGE2 is well established in MDSCs, the role of EP2/4 on human MDSCs or whether EP2/4 modulation can prevent MDSCs suppressive features upon exposure to tumor-derived PGE2 is poorly defined. In this study, using an in vitro model of human monocytic-MDSCs (M-MDSCs) we demonstrate that EP2 and EP4 signaling contribute to the induction of a pro-tumor phenotype and function on M-MDSCs. PGE2 signaling via EP2 and EP4 boosted M-MDSC ability to suppress T and NK cell responses. Combined EP2/4 blockade on M-MDSCs during PGE2 exposure prevented the occurrence of these suppressive features. Additionally, EP2/4 blockade attenuated the suppressive phenotype of M-MDSCs in a 3D coculture with colorectal cancer patient-derived organoids. Together, these results identify the role of tumor-derived PGE2 signaling via EP2 and EP4 in this human M-MDSC model, supporting the therapeutic value of targeting PGE2-EP2/4 axis in M-MDSCs to alleviate immunosuppression and facilitate the development of anti-tumor immunity.

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance.

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthetase (FPGS), which normally promotes intracellular accumulation and activity of natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. Here, we describe a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase and downstream one-carbon metabolism. Contrary to methotrexate, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folates for interaction with dihydrofolate reductase. We show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1, whereas FPGS-high CRC organoids are more sensitive to methotrexate. Our results argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, using a vulnerability created by polyglutamylation deficiency.

Therapeutic potential of dual CDK12 and 13 inhibition in colorectal cancer.

e15601 Background: To date, studies on cyclin-dependent kinase inhibitors in colorectal cancer (CRC), such as targeting CDK4/6 with Palbociclib, have shown little to no clinical benefit. However, tumor profiling of CRC has shown that mutations and amplifications in CDK 12 and 13 are prevalent in 5 to 10% of CRC patients, suggesting that CDK12/13 inhibition may be a potential target for patients with CRC. Methods: To study the effects of inhibiting CDK12 and 13 in CRC, a total of 11 patient-derived organoids (PDO) were first established from patients undergoing biopsy or resection of their primary or metastatic CRC. In vitro drug sensitivity assays were performed by treating each PDO with THZ531, a selective dual CDK12/13 inhibitor; 5-flurouracil, oxaliplatin and SN38 as a standard of care; or Palbociclib (CDK 4/6 inhibitor) at seven different drug concentrations (0.0064 μM - 100 μM) with five replicates at each concentration. Viability was assessed 72 hours following the addition of each drug using CellTiter-Glo (Promega) and IC50s were calculated. Results: Targeting CDK12/13 with THZ531 demonstrated dose dependent activity in the majority of CRC PDOs, with IC50 values ranging from 0.06 μM to 4.2 μM. In comparison, targeting CDK4/6 with Palbociclib was ineffective, with IC50 values ranging from 2.5 μM to 17 μM (p = 0.0003, 95% CI 3.7 to 10.4). Inhibition of CDK12/13 was also more effective than standard of care chemotherapy with either 5- FU or oxaliplatin (mean IC50 values were 4.2 μM and 21.4 μM for 5-fluorouracil and oxaliplatin, p < 0.05) and was comparable to irinotecan (mean IC50 was 0.07 μM for SN38). Finally, no significant correlation was found between sensitivity to THZ531 and microsatellite status, mutations in RAS and BRAF, or grade of the original patient tumor. Conclusions: We identify dual CDK12/13 inhibition as a promising therapy to treat CRC. Further studies will focus on characterizing mechanisms of action, evaluating efficacy and toxicity in vivo, and determining biomarkers of response based on actionable genomic alterations.

Translational relevance of SOS1 targeting for KRAS-mutant colorectal cancer.

It has been challenging to target mutant KRAS (mKRAS) in colorectal cancer (CRC) and other malignancies. Recent efforts have focused on developing inhibitors blocking molecules essential for KRAS activity. In this regard, SOS1 inhibition has arisen as an attractive approach for mKRAS CRC given its essential role as a guanine nucleotide exchange factor for this GTPase. Here, we demonstrated the translational value of SOS1 blockade in mKRAS CRC. We used CRC patient-derived organoids (PDOs) as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406. A combination of in silico analyses and wet lab techniques was utilized to define potential predictive markers for SOS1 sensitivity and potential mechanisms of resistance in CRC. RNA-seq analysis of CRC PDOs revealed two groups of CRC PDOs with differential sensitivities to SOS1 inhibitor BI3406. The resistant group was enriched in gene sets involving cholesterol homeostasis, epithelial-mesenchymal transition, and TNF-α/NFκB signaling. Expression analysis identified a significant correlation between SOS1 and SOS2 mRNA levels (Spearman's ρ 0.56, p < 0.001). SOS1/2 protein expression was universally present with heterogeneous patterns in CRC cells but only minimal to none in surrounding nonmalignant cells. Only SOS1 protein expression was associated with worse survival in patients with RAS/RAF mutant CRC (p = 0.04). We also found that SOS1/SOS2 protein expression ratio >1 by immunohistochemistry (p = 0.03) instead of KRAS mutation (p = 1) was a better predictive marker to BI3406 sensitivity of CRC PDOs, concordant with the significant positive correlation between SOS1/SOS2 protein expression ratio and SOS1 dependency. Finally, we showed that GTP-bound RAS level underwent rebound even in BI3406-sensitive PDOs with no change of KRAS downstream effector genes, thus suggesting upregulation of guanine nucleotide exchange factor as potential cellular adaptation mechanisms to SOS1 inhibition. Taken together, our results show that high SOS1/SOS2 protein expression ratio predicts sensitivity to SOS1 inhibition and support further clinical development of SOS1-targeting agents in CRC.

Abstract 199: Novel platform for automation of high throughput drug discovery using patient derived colorectal cancer organoids

Abstract Most potential oncology drugs fail at the later stages of the drug development pipeline and in clinical trials, despite having promising data for their efficacy in vitro. This high failure rate is partly due to insufficient predictive models being used to screen drug candidates in the early stages of drug discovery. As such, there is a need to develop and utilize more representative models that are amendable for efficient testing of drug efficacy to discover new therapeutic targets. 3D cell models, specifically patient-derived organoids (PDOs), offer a promising solution to this problem. Cells grown in 3D can better mimic cell-cell interactions and the tissue microenvironment, including cancer stem cell niches. Studies show that patients and their derived organoids respond similarly to drugs, suggesting the therapeutic value of using PDOs to improve therapeutic outcomes. However, challenges commonly associated with using these organoids, such as assay reproducibility, ability to scale up, and cost have limited their widespread adoption as a primary screening method in drug discovery. To address some of the hurdles associated with the use of PDOs in large scale screens, a semi-automated bioprocess has been developed for the controlled production of standardized PDOs at scale. PDOs cultured in the bioprocessor were uniform in size, show high viability and were produced in repeatable batches in an assay-ready format. In this study, patient-derived colorectal cancer (CRC) organoids were seeded in high density (96 or 384 well) microtiter plates manually. We also tested the feasibility of scaling up the use of these CRCs by using an automated liquid handler or a bioprinter to seed and culture the PDOs. CRC PDOs were treated with selected anti-cancer compounds at various concentrations. Compound effects were monitored over time using transmitted light imaging. For the analysis of organoid growth and development, a deep learning-based image segmentation model was developed to automate the segmentation of the organoids. Using this approach, we tracked the effects of the compounds on colorectal organoid size, morphology, texture, and additional morphological and phenotypic readouts. A viability assay was carried out using live/dead cell dyes and the PDOs were imaged in 3D on a high-content confocal imager. Out of the tested panel of known anti-cancer drugs, we found that PDOs treated with romidepsin and trametinib showed the most significant reduction in size, with a greater number of dead cells compared to the other compounds and controls. Overall, our results show the potential for the utility of PDOs in both precision medicine and high throughput drug discovery applications, when using automation with high-content imaging. Citation Format: Angeline Lim, Zhisong Tong, Prathyushakrishna Macha, Oksana Sirenkp. Novel platform for automation of high throughput drug discovery using patient derived colorectal cancer organoids [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 199.

Abstract 2383: Fluorescence lifetime imaging microscopy to assess drug response in patient-derived colorectal cancer organoids

Abstract Patient-derived tumor organoids (PDTOs) have been shown to be predictors of clinical response. While evidence suggests PDTOs are a more physiologically relevant cell model to use for drug discovery, challenges still exist to fully characterize their response to chemotherapies. Assays which attempt to quantify drug response in preclinical models are often end-point and cannot separate responses of heterogeneous cultures or capture dynamic information. Image based technologies attempt to counteract these shortcomings but often require fluorescent protein expression or cell staining to observe changes long-term. We utilized a label-free approach, fluorescence lifetime imaging microscopy (FLIM) and phasor analysis, to determine the metabolic state of individual PDTOs and correlate our findings to traditional cell viability assays (e.g., ATP measurements, phototoxic dyes) in response to drug perturbations. By measuring the fluorescence lifetime of NADH, an autofluorescent metabolite, we were able to quantify the metabolic signature of KRAS mutant (KRASMUT) colorectal cancer (CRC) PDTOs. The emission decay at each pixel was Fourier transformed and represented on a phasor plot. Pixels that had a shorter lifetime of NADH were indicative of glycolysis (GLY) while longer lifetimes indicated regions of oxidative phosphorylation (OXPHOS). PDTOs were treated with clinically-relevant drugs, SN-38 (an active metabolite of irinotecan), 5-FU, and cetuximab (CTX). FLIM images were taken after 6 and 72 hours of treatment. KRASMUT tumors do not respond to CTX, thus this was used as a negative control. Staurosporine (ST) was used as a positive control for cell death. A shift in metabolic signature towards OXPHOS in a dose-dependent manner was seen in PDTOs treated with ST or SN-38. These shifts correlated with cell death measured using the CellTiter Glo cell viability assay and confocal fluorescence images of PDTOs stained with a vital dye, DRAQ7. Changes in metabolism were seen within hours while DRAQ7 signal was detected after a few days. No changes in metabolic signature, cell viability, or DRAQ7 levels were seen in CTX treated PDTOs as expected. 5-FU was found to be an ineffective drug for the specific PDTO tested in our assay as shown by the lack of metabolic shifts compared to untreated PDTOs and no decrease in cell viability. Metabolic heterogeneity was also seen with FLIM, including distinguishing different cell types in co-cultures of PDTOs and cancer-associated fibroblasts (CAFs). We were able to separate the metabolic signatures of each cell type and determine that CAFs have a more GLY signature. Taken together, FLIM supported label-free imaging of multicellular 3D organoid models for the purposes of determining dynamic tumor drug response. We are adapting this workflow to a high-throughput multimodal imaging platform for drug screening to make more informed decisions on patient care. Citation Format: Emma Jane Fong, Mathias Bigger, Seungil Kim, Michael Doche, Scott Valena, Pratiksha Kshetri, Shannon Mumenthaler. Fluorescence lifetime imaging microscopy to assess drug response in patient-derived colorectal cancer organoids [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 2383.

Abstract 2574: An exon-skipping HDAC1 novel isoform promotes colorectal carcinogenesis

Abstract Introduction: In a recent profiling for alternative splicing variants in patient-derived colorectal cancer (CRC) organoids by long-read SMRT sequencing, we identified a hitherto undescribed in-frame spliced transcript of HDAC1 with exon 7 skipping (HDAC1-ΔEx7). This study aims to explore the potential role of HDAC1-ΔEx7 in colorectal carcinogenesis. Methods: Quantitative PCR was performed to evaluate the expression of HDAC1 canonical and HDAC1-ΔEx7 isoforms in a cohort of CRC patient (n=103). Functional biology of HDAC1-ΔEx7 was studied in colon organoids and in vivo model. RNA sequencing was carried out for mechanistic investigation. Results: HDAC1-ΔEx7, but not the full-length HDAC1, showed significant upregulations in CRC tumors compared to matching adjacent non-tumoral tissue (p &amp;lt; 0.0001). Change in HDAC1-ΔEx7 expression was not observed in gastric, hepatocellular and esophageal cancer, suggesting HDAC1-ΔEx7 might be exclusive to CRC. The increased HDAC1-ΔEx7 expressions could draw significant associations with inferior patients’ survivals and likely represent an independent prognostication tumor biomarker. Our functional studies highlighted that HDAC1 full-length and HDAC1-∆Ex7 had distinct biological consequences in CRC. Overexpression of HDAC1-ΔEx7 could induce malignant features of non-tumoral colon organoids, including transforming the single layer-luminal non-tumoral organoids into cancerous organoids with compact and multi-layer cell cluster structure, and increasing the ability of organoids’ long-term propagation. In addition, HDAC1-ΔEx7 could trigger anchor-independent spheroid formation in CRC cell line and protect against stress induced cell death. Investigation of metastatic potential of HDAC1 isoforms in BALB/c nude mice indicated that HDAC1-ΔEx7, but not canonical, substantially increased lung metastatic burden with respect to both lesions size and numbers. Our mechanistic investigation highlighted a unique role of HDAC1-ΔEx7 in protecting CRC against ferroptosis induced cell death. Overexpression of HDAC1-ΔEx7 significantly scavenged the lipid ROS induced by glutamate and inhibited ferroptosis hallmark genes expression. Re-expression of HDAC1-ΔEx7 upon CRISPR-based knocked out of endogenous HDAC1 could robustly rescue CRC against ferroptosis induced from Erastin. Transcriptome revealed that HDAC1-ΔEx7 overcomes ferroptosis through regulating ion homeostasis. Conclusion: Our study identified a novel exon 7 skipping isoform of HDAC1 in CRC and showed its oncogenic role in promoting CRC carcinogenesis through inferring resistance to ferroptosis. Citation Format: Guangzheng Deng, Yujuan Dong, Zhongxu Zhu, Yue Guo, Xin Wang, Simon Ng, Nathalie Wong. An exon-skipping HDAC1 novel isoform promotes colorectal carcinogenesis [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 2574.