Established Colorectal Cancer Research Articles

Efficacy assessment of a novel pan-RAS inhibitor in KRAS-mutant and wild type colorectal 3D bioprinted organoid tumor tissue.

91 Background: Colorectal cancer (CRC) is the third leading type of cancer worldwide, with ~150,000 new cases in the US annually and a grim 14% 5-year survival for patients diagnosed at a late stage. A lack of treatment options leads to persistently poor prognosis for patients with advanced stage disease. KRAS mutations are well known drivers of CRC and other GI cancers. Multiple KRAS mutations occur in CRC, including G12D (34%), G12V (21%), G13D (20%), G12C (8%), and others (18%). Existing KRAS-targeted therapies have limited use in CRC, underscoring the need for pan-RAS inhibitors in treating CRC and other RAS driven cancers. Objective: Assess activity of ADT-007, our pan-RAS inhibitor, on wild-type (WT) and KRAS-mutant 3D bioprinted organoid tumor (BOT) tissue using our high-throughput ex vivo platform. Methods: Using previously established bioprinting protocols, WT and mutant BOTs were printed with HT29 and HCT116 cells, respectively. HT29 is an established human WT CRC cell line with known sensitivity to proteosome and survivin inhibitors. HCT116 is a KRASG13D mutant human CRC cell line. 3 sets of BOTs were generated and acclimated for 24h. One set was treated for 72h with proteosome inhibitor Bortezomib, another with survivin inhibitor YM155, and the third with our novel pan-RAS inhibitor ADT-007. Dose response curves were generated from both conventional ATP luminescence readouts and high-content imaging. Results: BOT tissue microarchitecture was validated and >200 µm diffusion in BOTs was confirmed using high-content imaging. Differential response was quantified using Cell TiterGlo endpoint assay as well as advanced image processing of high-content live/dead nuclear stained images captured at multiple z-plains. ADT-007 IC50 was found to be substantially lower for mutant HCT116 compared to that for WT HT29 cell line BOTs, which was consistent with separately conducted in vitro and in vivo studies. Conclusions: A pan-RAS inhibitor, such as ADT-007 with high selectivity for cancer cells with activated RAS that is not limited to a specific KRAS mutant allele or RAS isozyme, could have broader use for CRC and other RAS-driven cancers. Further, due to their potential to replicate biophysical characteristics of a tumor and its microenvironment, BOT based precision and personalized medicine platforms can provide more accurate drug efficacy readout compared to in vitro cancer models.

Comparative Effectiveness and Cost-Effectiveness of Colorectal Cancer Screening With Blood-Based Biomarkers (Liquid Biopsy) vs Fecal Tests or Colonoscopy

Colorectal cancer (CRC) screening is highly effective but underused. Blood-based biomarkers (liquid biopsy) could improve screening participation. Using our established Markov model, screening every 3 years with a blood-based test that meets minimum Centers for Medicare & Medicaid Services' thresholds (CMSmin) (CRC sensitivity 74%, specificity 90%) was compared with established alternatives. Test attributes were varied in sensitivity analyses. CMSmin reduced CRC incidence by 40% and CRC mortality by 52% vs no screening. These reductions were less profound than the 68%-79% and 73%-81%, respectively, achieved with multi-target stool DNA (Cologuard; Exact Sciences) every 3 years, annual fecal immunochemical testing (FIT), or colonoscopy every 10 years. Assuming the same cost as multi-target stool DNA, CMSmin cost $28,500/quality-adjusted life-year gained vs no screening, but FIT, colonoscopy, and multi-target stool DNA were less costly and more effective. CMSmin would match FIT's clinical outcomes if it achieved 1.4- to 1.8-fold FIT's participation rate. Advanced precancerous lesion (APL) sensitivity was a key determinant of a test's effectiveness. A paradigm-changing blood-based test (sensitivity >90% for CRC and 80% for APL; 90% specificity; cost ≤$120-$140) would be cost-effective vs FIT at comparable participation. CMSmin could contribute to CRC control by achieving screening in those who will not use established methods. Substituting blood-based testing for established effective CRC screening methods will require higher CRC and APL sensitivities that deliver programmatic benefits matching those of FIT. High APL sensitivity, which can result in CRC prevention, should be a top priority for screening test developers. APL detection should not be penalized by a definition of test specificity that focuses on CRC only.

Abstract 6142: Interactions of processed meat and red meat intake with pathway-based polygenic risk scores for colorectal cancer: A novel approach for PRSs construction

Abstract Introduction: Colorectal cancer (CRC) is the third most common cancer, and second cause of cancer death worldwide. Established CRC risk factors (E) include high red meat and processed meat intake. Genome-wide association studies (GWAS) have reported over 200 genetic variants associated with CRC risk. We used functional annotation data to identify subsets of GWAS variants within known pathways and constructed corresponding pathway Polygenic Risk Scores (pPRS). We evaluated pPRS by E interactions to determine whether genes within specific pathways interacted with meat intake to impact CRC risk. Methods: A pooled sample of 54,531 CRC controls and 48,260 cases of European ancestry from 27 studies were analyzed. Study-specific quartiles for red and processed meat intake were generated through in-person interviews and structured self-administered questionnaires. Variants were imported to AnnoQ for annotation (n=203) and analyzed for overrepresentation in PANTHER-reported pathways with Fisher’s exact test. Standard approaches were used to compute polygenic risk score weights relating the 203 variants to CRC. The subset of weights corresponding to each of five pathways were then used to compute the corresponding pPRS. Covariate-adjusted logistic regression models evaluated pPRSxE interactions with red meat and processed meat intake. Results: A total of 34 unique variants were overrepresented in five pathways: Apoptosis signaling, Alzheimer disease-presenilin, Wnt-signaling, Gonadotropin-releasing hormone receptor, and TGF-beta signaling. We found a statistically significant interaction between TGF-beta-pPRS and red meat intake (p = 0.0012). Stratified analyses reported a dose-response trend in the red meat and CRC risk association, with decreasing estimates of red meat and CRC risk association, comparing first to fourth quartiles of meat intake, with increasing quartiles of TGF-beta-pPRS: 13% (Q1), 12% (Q2), 7% (Q3), and 6% (Q4). This association remained significant after adjustment for all other variants (p = 0.0013), and all other pPRS with variants that did not overlap with the TGF-beta-pPRS (p = 0.0012). Independent GxE interaction models for individual variants that were included in the TGF-beta pathway showed significant interactions with red meat for rs2337113 (intron SMAD7 gene, Chr18), and rs2208603 (intergenic region BMP5, Chr6) (p = 0.013 & 0.0108, respectively). We did not find significant pPRS x red meat interactions for the other four pathways or with any pPRS x processed meat. Conclusion: This pathway-based interaction analysis revealed a statistical interaction between SNPs in the TGF-beta pathway and red meat consumption that impacts CRC risk. These findings shed light into the possible mechanistic link between CRC risk and red meat consumption. Citation Format: Joel Sanchez Mendez, Mariana C. Stern, Yubo Fu, John Morrison, Juan P. Lewinger, Eric Kawaguchi, Bryan Queme, Huaiyu Mi, Flora Qu, Ulrike Peters, Li Hsu, Stephen B. Gruber, Li Li, Michelle Cotterchio, Loic Le Marchand, Andrew J. Pellat, Elizabeth A. Platz, W James Gauderman. Interactions of processed meat and red meat intake with pathway-based polygenic risk scores for colorectal cancer: A novel approach for PRSs construction [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 6142.

A GENOME-WIDE ASSOCIATION STUDY IN A ROMANIAN COLORECTAL CANCER COHORT IDENTIFIES GENETIC MARKERS ASSOCIATED WITH SUSCEPTIBILITY TO SARS-COV-2 INFECTIONS

Colorectal cancer ranks second in the incidence of cancer deaths worldwide. Our objectives are to identify genetic markers associated with colorectal cancer susceptibility using a genome-wide association study and to evaluate the identified genetic markers in the context of associated SARS-CoV-2 infections in the Romanian population. The study population consisted of 576 unrelated histopathologically confirmed colorectal cancer (CRC) cases and 1,069 controls consisting of patients admitted for medical conditions, excluding cancer. The DNA was extracted from buccal swab samples and analyzed by deCODE Genetics (Reykjavik, Iceland). We used the NHGRI catalog database to identify 85 unique variants from a systematic literature review for variants associated with CRC. Two of the tested variants in the Romanian GWAS reached genomewide significance (p-value lower than 5 × 10-8), but one of them did not match the other criteria. The top three markers were located on chr3, and the following two were located on chr2 and chr12, respectively. The Romanian GWAS results for CRC were compared to established markers linked to the measurement of COVID-19 symptoms. This allowed us to identify loci that may be useful in the future for assessing prognosis, treatment, and outcomes. We confirmed that established colorectal cancer SNPs are linked to colorectal cancer risk in the Romanian population while also examining the potential genetic connections between colorectal cancer predisposition and SARS-CoV-2 infection and symptoms.

Salicylate induces AMPK and inhibits c-MYC to activate a NRF2/ARE/miR-34a/b/c cascade resulting in suppression of colorectal cancer metastasis

Aspirin and its active metabolite salicylate have emerged as promising agents for the chemoprevention of colorectal cancer (CRC). Moreover, aspirin suppresses the progression of established CRCs. However, the underlying molecular mechanisms are not completely understood. Here we found that salicylate induces the expression of the miR-34a and miR-34b/c genes, which encode tumor suppressive microRNAs, in a p53-independent manner. Salicylate activated AMPK, thereby activating NRF2, which directly induced miR-34a/b/c expression via ARE motifs. In addition, salicylate suppressed c-MYC, a known repressor of NRF2-mediated transactivation, via activating AMPK. The suppression of c-MYC by salicylate was necessary for NRF2-mediated activation of miR-34a/b/c. Inactivation of miR-34a/b/c largely abrogated the inhibitory effects of salicylate on migration, invasion and metastasis formation by CRC cells. In the future, aspirin and its derivates may be used therapeutically to activate miR-34a and miR-34b/c in tumors that have lost p53.

Luciferase Expressing Preclinical Model Systems Representing the Different Molecular Subtypes of Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease. More insight into the biological diversity of CRC is needed to improve therapeutic outcomes. Established CRC cell lines are frequently used and were shown to be representative models of the main subtypes of CRC at the genomic and transcriptomic level. In the present work, we established stable, luciferase expressing derivatives from 10 well-established CRC cell lines, generated spheroids and subcutaneous xenograft tumors in nude mice, and performed comparative characterization of these model systems. Transcriptomic analyses revealed the close relation of cell lines with their derived spheroids and xenograft tumors. The preclinical model systems clustered with patient tumor samples when compared to normal tissue thereby confirming that cell-line-based tumor models retain specific characteristics of primary tumors. Xenografts showed different differentiation patterns and bioluminescence imaging revealed metastatic spread to the lungs. In addition, the models were classified according to the CMS classification system, with further sub-classification according to the recently identified two intrinsic epithelial tumor cell states of CRC, iCMS2 and iCMS3. The combined data showed that regarding primary tumor characteristics, 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do. Furthermore, we set up a bioluminescence-based spheroid cytotoxicity assay in order to be able to perform dose-response relationship studies in analogy to typical monolayer assays. Applying the established assay, we studied the efficacy of oxaliplatin. Seven of the ten used cell lines showed a significant reduction in the response to oxaliplatin in the 3D-spheroid model compared to the 2D-monolayer model. Therapy studies in selected xenograft models confirmed the response or lack of response to oxaliplatin treatment. Analyses of differentially expressed genes in these models identified CAV1 as a possible marker of oxaliplatin resistance. In conclusion, we established a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC, which can be used in preclinical research applications.

Stool DNA testing for early detection of colorectal cancer: systematic review using the HTA Core Model® for Rapid Relative Effectiveness Assessment.

Stool DNA testing for early detection of colorectal cancer (CRC) is a non-invasive technology with the potential to supplement established CRC screening tests. The aim of this health technology assessment was to evaluate effectiveness and safety of currently CE-marked stool DNA tests, compared to other CRC tests in CRC screening strategies in an asymptomatic screening population. The assessment was carried out following the guidelines of the European Network for Health Technology Assessment (EUnetHTA). This included a systematic literature search in MED-LINE, Cochrane and EMBASE in 2018. Manufacturers were asked to provide additional data. Five patient interviews helped assessing potential ethical or social aspects and patients' experiences and preferences. We assessed the risk of bias using QUADAS-2, and the quality of the body of evidence using GRADE. We identified three test accuracy studies, two of which investigated a multitarget stool DNA test (Cologuard®, compared fecal immunochemical test (FIT)) and one a combined DNA stool assay (ColoAlert®, compared to guaiac-based fecal occult blood test (gFOBT), Pyruvate Kinase Isoenzyme Type M2 (M2-PK) and combined gFOBT/M2-PK). We found five published surveys on patient satisfaction. No primary study investigating screening effects on CRC incidence or on overall mortality was found. Both stool DNA tests showed in direct comparison higher sensitivity for the detection of CRC and (advanced) adenoma compared to FIT, or gFOBT, respectively, but had lower specificity. However, these comparative results may depend on the exact type of FIT used. The reported test failure rates were higher for stool DNA testing than for FIT. The certainty of evidence was moderate to high for Cologuard® studies, and low to very low for the ColoAlert® study which refers to a former version of the product and yielded no direct evidence on the test accuracy for ad-vanced versus non-advanced adenoma. ColoAlert® is the only stool DNA test currently sold in Europe and is available at a lower price than Cologuard®, but reliable evidence is lacking. A screening study including the current product version of ColoAlert® and suitable comparators would, therefore, help evaluate the effectiveness of this screening option in a European context.

Colonoscopy Needs for Implementation of a Colorectal Cancer Screening Program in Ukraine.

In Ukraine, there is no established colorectal cancer screening program. We aimed to project the number of screening colonoscopies needed for implementation of various CRC screening strategies in Ukraine. We modified a previously developed Markov microsimulation model to reflect the natural history of adenoma and CRC progression among average-risk 50-74-year-olds. We simulated colonoscopies needed for the following screening strategies: no screening, fecal occult blood test yearly, FOBT yearly with flexible sigmoidoscopy every 5years, FS every 5years, fecal immunohistochemistry test (FIT) yearly, or colonoscopy every 10years. Assuming 80% screening adherence, we estimated colonoscopies required at 1 and 5years depending on the implementation rate. In one-way sensitivity analyses, we varied implementation rate, screening adherence, sensitivity, and specificity. Assuming an 80% screening adherence and complete implementation (100%), besides a no screening strategy, the fewest screening colonoscopies are needed with an FOBT program, requiring on average 6,600 and 26,800 colonoscopies per 100,000 persons at 1 and 5years post-implementation, respectively. The most screening colonoscopies are required with a colonoscopy program, requiring on average 76,600 and 101,000 colonoscopies per 100,000 persons at 1 and 5years post-implementation, respectively. In sensitivity analyses, the biggest driver of number of colonoscopies needed was screening adherence. The number of colonoscopies needed and therefore the potential strain on the healthcare system vary substantially by screening test. These findings can provide valuable information for stakeholders on equipment needs when implementing a national screening program in Ukraine.

Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66bhighCD11bhighCD62Llow phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86lowCD163+CD206+ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.

The Mitochondrial Disruptor Devimistat (CPI-613) Synergizes with Genotoxic Anticancer Drugs in Colorectal Cancer Therapy in a Bim-Dependent Manner.

Colorectal cancer is one of the most frequent tumor entities, with an increasing incidence and mortality in younger adults in Europe and the United States. Five-year survival rates for advanced colorectal cancer are still low, highlighting the need for novel targets in colorectal cancer therapy. Here, we investigated the therapeutic potential of the compound devimistat (CPI-613) that targets altered mitochondrial cancer cell metabolism and its synergism with the antineoplastic drugs 5-fluorouracil (5-FU) and irinotecan (IT) in colorectal cancer. Devimistat exerted a comparable cytotoxicity in a panel of established colorectal cancer cell lines and patient-derived short-term cultures independent of their genetic and epigenetic status, whereas human colonic epithelial cells were more resistant, indicating tumor selectivity. These findings were corroborated in intestinal organoid and tumoroid models. Mechanistically, devimistat disrupted mitochondrial membrane potential and severely impaired mitochondrial respiration, resulting in colorectal cancer cell death induction independent of p53. Combination treatment of devimistat with 5-FU or IT demonstrated synergistic cell killing in colorectal cancer cells as shown by Combenefit modeling and Chou-Talalay analysis. Increased cell death induction was revealed as a major mechanism involving downregulation of antiapoptotic genes and accumulation of proapoptotic Bim, which was confirmed by its genetic knockdown. In human colorectal cancer xenograft mouse models, devimistat showed antitumor activity and synergized with IT, resulting in prolonged survival and enhanced therapeutic efficacy. In human tumor xenografts, devimistat prevented IT-triggered p53 stabilization and caused synergistic Bim induction. Taken together, our study revealed devimistat as a promising candidate in colorectal cancer therapy by synergizing with established antineoplastic drugs in vitro and in vivo.

TP10.2.11The consequences of COVID-19 on the colorectal red-flag cancer pathway- A 3 month review 2019 to 2020

AimThe COVID-19 pandemic is an evolving healthcare challenge introducing greater burden on existing resources. With 1,100 people in Northern Ireland diagnosed with colorectal cancer (CRC) per annum, concerns over disruption of cancer services and secondary consequences have been highlighted. We aim to evaluate the impact of COVID-19 on the CRC red flag pathway in comparison to the pre-COVID era.MethodsTwo comparative data sets were compiled through retrospective analysis of red-flag colorectal referrals over a 3-month period for both April to June 2019 and 2020. A comprehensive review of each patient’s electronic care record and medical notes was completed. Patient demographics, co-morbidity, referral information, time to hospital appointment and investigation modality were documented. For patients identified with CRC the stage and time to first definitive treatment was documented.ResultsA total of 47 CRCs were identified from both red-flag referral groups; 25 CRCs 2019 compared to 22 in 2020. Median age at time of referral was 79 years in 2019 compared to 71 years in 2020. Time to outpatient review was significantly less during 2020 compared to 2019; 16 days and 31 days respectively (p < 0.05). Time to first treatment was 103 days 2019 compared to 75 days 2020 (p < 0.05). Advanced diagnostic stage or increased number of emergency hospital presentations in the COVID-19 period was not demonstrated.ConclusionDespite disruption of established colorectal cancer services during the COVID-19 pandemic, we demonstrated patients waited less time to outpatient review and intervention. With comparative cases of CRC to the pre-COVID era diagnosed.

Identification of histone methyltransferase NSD2 as an important oncogenic gene in colorectal cancer

Colorectal cancer (CRC) is the second common cause of cancer-related human mortalities. Dysregulation of histone 3 (H3) methylation could lead to transcriptional activation of multiple oncogenes, which is closely associated with CRC tumorigenesis and progression. Nuclear receptor-binding SET Domain protein 2 (NSD2) is a key histone methyltransferase catalyzing histone H3 lysine 36 dimethylation (H3K36me2). Its expression, the potential functions, and molecular mechanisms in CRC are studied here. Gene Expression Profiling Interactive Analysis (GEPIA) bioinformatics results showed that the NSD2 mRNA expression is elevated in both colon cancers and rectal cancers. Furthermore, NSD2 mRNA and protein expression levels in local colon cancer tissues are significantly higher than those in matched surrounding normal tissues. In primary human colon cancer cells and established CRC cell lines, shRNA-induced silencing or CRISPR/Cas9-induced knockout of NSD2 inhibited cell viability, proliferation, cell cycle progression, migration, and invasion. Furthermore, NSD2 shRNA or knockout induced mitochondrial depolarization, DNA damage, and apoptosis in the primary and established CRC cells. Contrarily, ectopic NSD2 overexpression in primary colon cancer cells further enhanced cell proliferation, migration, and invasion. H3K36me2, expressions of multiple oncogenes (ADAM9, EGFR, Sox2, Bcl-2, SYK, and MET) and Akt activation were significantly decreased after NSD2 silencing or knockout in primary colon cancer cells. Their levels were however increased after ectopic NSD2 overexpression. A catalytic inactive NSD2 (Y1179A) also inhibited H3K36me2, multiple oncogenes expression, and Akt activation, as well as cell proliferation and migration in primary colon cancer cells. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD2 shRNA largely inhibited primary colon cancer cell xenograft growth in nude mice. Together, NSD2 exerted oncogenic functions in CRC and could be a promising therapeutic target.

Caspase-cleavable peptide-doxorubicin conjugate in combination with CD47-antagonizing nanocage therapeutics for immune-mediated elimination of colorectal cancer

Here we report a novel combination of a caspase-cleavable peptide-doxorubicin conjugate (MPD-1) with CD47-antagonizing nanocage therapeutics for the treatment of microsatellite-stable (MSS) colorectal cancer (CRC). MPD-1 (i) upregulated markers of immunogenic cell death (ICD) in tumor, and increased co-stimulatory markers on dendritic cells (DCs), (ii) enhanced CD8+ T cell infiltration and antigen presenting cell (APC) activation, and (iii) showed negligible off-target immune-related toxicity compared to free dox. Then, the CD47 antagonist FS nanocage, a SIRPα-expressing ferritin nanocage, was co-administered with MPD-1 that resulted in 95.2% (p < 0.001) tumor growth inhibition in an established CRC model. T cell-mediated elimination of tumors was also confirmed by the tumor-specific activation of T cells detected by IFNγ and tumor-free mice were observed (95%) that bared a memory response when re-challenged. The strategically developed MPD-1 is an ideal adjuvant to immunotherapy and the combination with FS nanocage triggers potent immunity against MSS CRC. In summary, we present an approach to initiate and stimulate immune-mediated eradication of cancer cells using synergistic immunogenic agents targeting the MSS CRC.

Abstract 2880: CD36, a fatty acid translocase, promotes metastasis in CRC

Abstract Altered fatty acid metabolism, a hallmark of cancer, is an attractive target for therapeutic intervention. Fatty acid translocase (CD36), a multifunctional glycoprotein, has an important role in fatty acid metabolism: it serves as a fatty acid transporter. We have previously shown the importance of CD36 in promotion of colorectal cancer (CRC) cell proliferation and tumorigenesis and its role in compensation of de novo fatty acid synthesis inhibition. CD36 also plays important roles in migration, invasion and metastasis in gastric, ovarian and oral carcinomas. Cell lines with higher metastatic potential express higher levels of CD36; however, the role of CD36 in CRC metastasis has not been previously studied. The purpose of our study is (i) to determine the functional effect of CD36 on CRC metastasis, and (ii) to delineate the mechanistic pathways in which CD36 may regulate CRC metastasis. Methods: In this study we utilized HCT116 and HT29 CRC cell lines. The HT29 LuM3-GFP-Luc cell line was established from HT29 cells and has an increased propensity to metastasize to mouse lungs. The increase in metastatic capacity of HT29 LuM3 is associated with an increase in the expression of CD36. We utilized parental HT29-GFP-Luc and HT29 LuM3-GFP-Luc to overexpress and knockdown CD36, respectively, and we used a CD36 neutralizing antibody to block the activity of CD36 in HCT116 and HT29 LuM3 cells. To measure colonization, migration and invasion in vitro, we used soft agar colony formation assays, a wound healing assay, and spheroid and trans-well invasion assays. In vivo, we utilized the tail-vein injection model to study tumor cell colonization in mouse lungs, and the cecum injection model to study liver metastasis from the primary tumor. We analyzed samples with qRT-PCR, western blot and immunohistochemistry. Results: Knockdown of CD36 significantly reduced CRC cell invasion and colony formation in HCT116 and HT29 LuM3 in vitro. CRC cell invasion and colony formation increased with overexpression of CD36 in HCT116 cells. Overexpression of CD36 in established CRC cell lines increased markers of survival and invasion, such as phospho-Akt, P-Met, and MMP28. Most importantly, knockdown of CD36 in HT29 LuM3-GFP-Luc cells significantly reduced lung metastasis in vivo. Conclusions: CRC cells that have a higher propensity to initiate metastasis in vivo express higher levels of CD36. We showed that this upregulation of CD36 represents a unique advantage for CRC cells to initiate tumor colonies, migrate and invade both in vitro and in vivo. Thus, our study highlights the potential of CD36 as a viable target for metastatic disease in CRC patients. Citation Format: James Michael Drury, Piotr Rychahou, Heidi L. Weiss, Yekaterina Y. Zaytseva. CD36, a fatty acid translocase, promotes metastasis in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2880.

Abstract 115: Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment

Abstract Neutrophil extracellular traps (NETs) have recently been implicated in the growth of tumors, however their impact on immune cells within the tumor microenvironment (TME) remain incompletely understood. We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. We performed liver Ischemia/Reperfusion (I/R) in an established colorectal cancer metastasis model to induce NET formation in the TME. In this NET-rich TME we discovered elevated levels of phenotypically and functionally exhausted T cells. We demonstrated that the majority of CD4 and CD8 T cells are not effector cells, but rather express multiple inhibitory receptors and are highly dysfunctional, with reduced cytokine production and proliferative capacity. This effect was limited to the T cells present the the NET-rich TME in comparison to T cells isolated from the spleen. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion; an effect abrogated by using PD-L1 KO NETs. This exhausted state was directly linked to mitochondrial dysfunction in the T cells. Furthermore, NETs were targeted in vivo by treating mice with DNAse or anti-PD-L1 at time of I/R. Treatment caused decreased metastatic burden, decreased NET formation and higher levels of functioning non-exhausted T cells. Conversely, injecting NETs in tumors led to acceleration of tumor growth and T cell exhaustion in the TME. In addition, we measured sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. High levels of both markers were significantly associated with decreased disease free survival. Neutrophils isolated from patients after surgery were primed to form NETs that caused a similar exhaustion and dysfunction of human CD4 and CD8 T cells. Our data thus reveal that NETs have the capability of suppressing T cell responses and promoting tumor growth. This mechanism of T cell suppression by NETs represents a viable target for sustaining immune competence within the TME. Citation Format: Christof Kaltenmeier, Hamza Yazdani, Samer Tohme. Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 115.

Circulating B-vitamin biomarkers and B-vitamin supplement use in relation to quality of life in patients with colorectal cancer: results from the FOCUS consortium

B vitamins have been associated with the risk and progression of colorectal cancer (CRC), given their central roles in nucleotide synthesis and methylation, yet their association with quality of life in established CRC is unclear. To investigate whether quality of life 6 months postdiagnosis is associated with: 1) circulating concentrations of B vitamins and related biomarkers 6 months postdiagnosis; 2) changes in these concentrations between diagnosis and 6 months postdiagnosis; 3) B-vitamin supplement use 6 months postdiagnosis; and 4) changes in B-vitamin supplement use between diagnosis and 6 months postdiagnosis. We included 1676 newly diagnosed stage I-III CRC patients from 3 prospective European cohorts. Circulating concentrations of 9 biomarkers related to the B vitamins folate, riboflavin, vitamin B6, and cobalamin were measured at diagnosis and 6 months postdiagnosis. Information on dietary supplement use was collected at both time points. Health-related quality of life (global quality of life, functioning scales, and fatigue) was assessed by the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire 6 months postdiagnosis. Confounder-adjusted linear regression analyses were performed, adjusted for multiple testing. Higher pyridoxal 5'-phosphate (PLP) was cross-sectionally associated with better physical, role, and social functioning, as well as reduced fatigue, 6 months postdiagnosis. Associations were observed for a doubling in the hydroxykynurenine ratio [3-hydroxykynurenine: (kynurenic acid+xanthurenic acid+3-hydroxyanthranilic acid+anthranilic acid); an inverse marker of vitamin B6] and both reduced global quality of life (β = -3.62; 95% CI: -5.88, -1.36) and worse physical functioning (β = -5.01; 95% CI: -7.09, -2.94). Dose-response relations were observed for PLP and quality of life. No associations were observed for changes in biomarker concentrations between diagnosis and 6 months. Participants who stopped using B-vitamin supplements after diagnosis reported higher fatigue than nonusers. Higher vitamin B6 status was associated with better quality of life, yet limited associations were observed for the use of B-vitamin supplements. Vitamin B6 needs further study to clarify its role in relation to quality of life.

Dietary intake of branched-chain amino acids and survival after colorectal cancer diagnosis.

Branched-chain amino acids (BCAAs), including leucine, isoleucine and valine, may potentially influence cancer progression by various mechanisms including its role in insulin resistance. However, the association of BCAAs with survival among patients with established colorectal cancer (CRC) remains unclear. We evaluated the associations between postdiagnostic BCAA intake with CRC-specific mortality and overall mortality among 1674 patients with nonmetastatic CRC in the Nurses' Health Study and the Health Professionals Follow-up Study. Patients completed a validated food frequency questionnaire. Multivariable hazard ratios (HRs) were calculated using Cox proportional-hazards regression model after adjustment for tumor characteristics and potential confounding factors. Comparing the highest with the lowest quartile intake of postdiagnostic total BCAA, the multivariable HRs were 1.18 (95% confidence interval [CI], 0.75-1.85, P for trend = .46 across quartiles) for CRC-specific mortality and 1.30 (95% CI, 1.01-1.69, P for trend = .04) for all-cause mortality. The multivariable HRs (the highest vs the lowest quartile) for all-cause mortality were 1.33 (95% CI, 1.03-1.73, Ptrend = .02) for valine, 1.28 (95% CI, 0.99-1.66, P for trend = .05) for leucine and 1.25 (95% CI, 0.96-1.61, P for trend = .06) for isoleucine. No statistically significant associations with each of the BCAA intake were observed for CRC-specific mortality (all P for trend > .30). Our findings suggest positive associations between higher intake of dietary BCAAs and risk of all-cause mortality in CRC patients. These findings need to be confirmed and potential mechanisms underlying this association need to be elucidated.

S1P Lyase siRNA Dampens Malignancy of DLD-1 Colorectal Cancer Cells.

Sphingosine-1-phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine-1-phosphate-degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD-1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell-cell-adhesion through upregulation of E-cadherin and formation of cadherin-actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.

Abstract 1494: Etiologies of patient-derived colorectal cancer organoid growth heterogeneity across multiple patient samples and culture conditions

Abstract Background: Patient-derived cancer organoids (PDCOs) are emerging as an in vitro model to recapitulate the molecular and phenotypic features of colorectal cancer (CRC). Heterogeneity in established CRC PDCOs has been observed in the underlying molecular profiles and growth characteristics at the individual organoid level. Here, we present a dedicated assessment of individual organoid growth as a function of experimental culture parameters. Methods: CRC PDCO cultures were established from patient biopsy/resection specimens following patient consent to an IRB-approved protocol. Growth was assessed by relative change in diameter at 48h. Baseline size was compared to relative growth at 48h using coefficient of determination (R). Interquartile sets were compared by effect size of Glass's Delta (GΔ) to compare change difference in average growth normalized to standard deviation and defined with significance 1.0. Impact of density was assessed by manual count of spheroids and lines plated across varied densities and compared to 48h relative growth. Growth rates were compared as both absolute and relative passage number. Results: 22 unique cultures were established from fresh tissue and representative of CRC including pathologic alterations in APC, KRAS, NRAS, BRAF, PIK3CA TP53, MTOR and PTEN. Pairwise spheres were assessed at baseline and 48h for analysis (n=1714) with mean relative growth rate of 27.1% (range -40.0, 156.2%). Baseline size did not predict relative growth at 48h (R=0.023) with insignificant interquartile effect size [0.10, 0.07, 0.32]. Replicates (n=63) across a range of passages [1, 36] including line-specific relative passage number [0-14] did not predict change in relative growth (R&amp;lt;0.001) with insignificant interquartile effect size [-0.02, 0.15, 0.19]. Fields of view (n=135) were assessed for absolute sphere number [1, 72]. Increased sphere density across cultures also did not predict change in relative growth (R=0.002) with insignificant interquartile effect size [-0.29, -0.32, -0.29]. Two individual cultures were assessed for the impact of density on respective growth without significance at relative plating ratios of 1:5 (GΔ=-0.52, 0.42), 1:10 (GΔ=-0.63, 0.07), and 1:50 (GΔ=-0.40, 0.12). Conclusions: Following culture maturation, CRC PDCOs have heterogeneous growth rates at the organoid level. These studies demonstrate that the growth rate is independent of baseline organoid size, passage number, or culture plating density. Understanding the effect of culture variation helps to define meaningful population effects in response and resistance to therapy and supports the translation of this technology as a future predictive biomarker. Citation Format: Aishwarya Sunil, Jeremy D. Kratz, Sarbjeet K. Makkar, Suhjah Rehman, Amani A. Gillette, Katherine A. Johnson, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Melissa C. Skala, Dustin A. Deming. Etiologies of patient-derived colorectal cancer organoid growth heterogeneity across multiple patient samples and culture conditions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1494.

Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells.

Fatty acid synthase, a key enzyme of de novo lipogenesis, is an attractive therapeutic target in cancer. The novel fatty acid synthase inhibitor, TVB-3664, shows anti-cancer activity in multiple cancers including colorectal cancer; however, it is unclear whether uptake of exogeneous fatty acids can compensate for the effect of fatty acid synthase inhibition. This study demonstrates that inhibition of fatty acid synthase selectively upregulates fatty acid translocase (CD36), a fatty acid transporter, in multiple colorectal cancer models including colorectal cancer cells with shRNA mediated knockdown of fatty acid synthase and genetically modified mouse tissues with heterozygous and homozygous deletion of fatty acid synthase. Furthermore, human colorectal cancer tissues treated with TVB-3664 show a significant and selective upregulation of CD36 mRNA. shRNA-mediated knockdown of CD36 and inhibition of CD36 via sulfosuccinimidyl oleate, a chemical inhibitor of CD36, decreased cell proliferation in vitro and reduced tumor growth in subcutaneous xenograft models. Isogenic cell populations established from patient derived xenografts and expressing high levels of CD36 show a significantly increased ability to grow tumors in vivo. The tumor-promoting effect of CD36 is associated with an increase in the levels of pAkt and survivin. Importantly, combinatorial treatment of primary and established colorectal cancer cells with TVB-3664 and sulfosuccinimidyl oleate shows a synergistic effect on cell proliferation. In summary, our study demonstrates that upregulation of CD36 expression is a potential compensatory mechanism for fatty acid synthase inhibition and that inhibition of CD36 can improve the efficacy of fatty acid synthase-targeted therapy.