Colorectal Carcinoma Model Research Articles

Abstract 3050: Multimeric IgM antibodies targeting DR5 are potent and rapid inducers of tumor cell apoptosis and cell death in vitro and in vivo

Abstract The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, death receptor 4 (DR4) and death receptor 5 (DR5) are attractive anti-tumor targets because of their ability to directly induce apoptosis in cancer cells. Though several agonistic IgG antibodies targeting DR5 have demonstrated evidence of preclinical efficacy, little evidence of clinical efficacy was observed likely due to insufficient receptor crosslinking in the tumor microenvironment. We have developed a novel multivalent IgM antibody targeting DR5 that effectively clusters the receptor and rapidly induces apoptosis and tumor cell cytotoxicity. In Colo205 colorectal tumor cells, anti-DR5 IgM induced caspase activation was detected within 30 minutes and by 2 hours nearly all cells had undergone apoptosis, demonstrating a more rapid and greater magnitude of apoptotic induction than anti-DR5 IgG. In an in vitro cytotoxicity screen of solid and hematologic tumor cell lines, an IgM antibody targeting DR5 ranged from 100-fold to greater than 10,000-fold more potent compared to IgG targeting DR5, and IgM was potent on cell lines resistant to IgG crosslinking mediated cell death. In vivo, anti-DR5 IgM induced tumor eradication in the Colo205 model, tumor regression in the IgG-resistant colorectal HCT15 model, and significantly extended overall survival in the B-cell leukemia model Nalm-6 even though dosing was only within the first week of study. In all in vivo studies the IgM was significantly more efficacious than IgG targeting DR5. The DR5 targeting IgM antibody was also efficacious in large established Colo205 tumors up to 600 mm3 in volume and in colorectal patient-derived xenograft (PDX) models as well. When administered in conjunction with standard of care chemotherapy Irinotecan, anti-DR5 IgM induced durable tumor regression in the HCT15 colorectal carcinoma model with 14 out of 30 animals from 2 separate studies tumor free at the end of study (day 71); no animals dosed with IgG were tumor free. In a less sensitive pancreatic model BxPC3, combination of standard of care Gemcitabine with anti-DR5 IgM resulted in an additive effect, while the IgG plus Gemcitabine efficacy was more modest and not statistically significant compared to Gemcitabine alone. Taken together, these results demonstrate that efficient IgM clustering of DR5 is significantly more potent in vitro and in vivo than IgGs and support the development of a human IgM antibody therapeutic targeting DR5 with the potential to treat both solid and hematologic tumors. Citation Format: Beatrice Wang, Tasnim Kothambawala, Ling Wang, Avneesh Saini, Ramesh Baliga, Angus Sinclair, Bruce Keyt. Multimeric IgM antibodies targeting DR5 are potent and rapid inducers of tumor cell apoptosis and cell death in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3050.

Vimentin activation in early apoptotic cancer cells errands survival pathways during DNA damage inducer CPT treatment in colon carcinoma model

Epithelial to mesenchymal transitions (EMT) is a preparatory process for cancer cells to attain motility and further metastasis to distant sites. Majority of DNA damaging drugs have shown to develop EMT as one of the major mechanisms to attain drug resistance. Here we sought to understand the resistance/survival instincts of cancer cells during initial phase of drug treatment. We provide a tangible evidence of stimulation of EMT factors in Apc knockout colorectal carcinoma model. Our results implied that CPT-treated Apc knockout cohorts depicted increased pro-invasive and pro-survival factors (Vimentin/pser38Vimentin & NFκB). Moreover, by cell sorting experiment, we have observed the expression of Vimentin in early apoptotic cells (AnnexinV positive) from 36 to 48 h of CPT treatment. We also observed the expression of chimeric Sec-AnnexinV-mvenus protein in migrated cells on transwell membrane recapitulating signatures of early apoptosis. Notably, induction of Vimentin-mediated signaling (by CPT) delayed apoptosis progression in cells conferring survival responses by modulating the promoter activity of NFκB. Furthermore, our results unveiled a novel link between Vimentin and ATM signaling, orchestrated via binding interaction between Vimentin and ATM kinase. Finally, we observed a significant alteration of crypt-villus morphology upon combination of DIM (EMT inhibitor) with CPT nullified the background EMT signals thus improving the efficacy of the DNA damaging agent. Thus, our findings revealed a resistance strategy of cancer cells within a very initial period of drug treatment by activating EMT program, which hinders the cancer cells to achieve later phases of apoptosis thus increasing the chances of early migration.

Flexicaulin A, An ent-Kaurane Diterpenoid, Activates p21 and Inhibits the Proliferation of Colorectal Carcinoma Cells through a Non-Apoptotic Mechanism.

Natural products, explicitly medicinal plants, are an important source of inspiration of antitumor drugs, because they contain astounding amounts of small molecules that possess diversifying chemical entities. For instance, Isodon (formerly Rabdosia), a genus of the Lamiaceae (formerly Labiatae) family, has been reported as a rich source of natural diterpenes. In the current study, we evaluated the in vitro anti-proliferative property of flexicaulin A (FA), an Isodon diterpenoid with an ent-kaurane structure, in human carcinoma cells, by means of cell viability assay, flow cytometric assessment, quantitative polymerase chain reaction array, Western blotting analysis, and staining experiments. Subsequently, we validated the in vivo antitumor efficacy of FA in a xenograft mouse model of colorectal carcinoma. From our experimental results, FA appears to be a potent antitumor molecule, since it significantly attenuated the proliferation of human colorectal carcinoma cells in vitro and restricted the growth of corresponsive xenograft tumors in vivo without causing any adverse effects. Regarding its molecular mechanism, FA considerably elevated the expression level of p21 and induced cell cycle arrest in the human colorectal carcinoma cells. While executing a non-apoptotic mechanism, we believe the antitumor potential of FA opens up new horizons for the therapy of colorectal malignancy.

Toward the Optimization of Click-Mediated Pretargeted Radioimmunotherapy.

Pretargeted radioimmunotherapy (PRIT) based on the inverse electron demand Diels-Alder reaction has shown promise in murine models of disease, yet the radiation dosimetry of this approach must be optimized to make it a viable clinical option. To this end, we have leveraged two recent developments in pretargeted imaging-dendritic scaffolds and masking agents-to improve the dosimetric profile of a proof-of-concept PRIT system that is based on the huA33 antibody, a 177Lu-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz), and a mouse model of A33 antigen-expressing colorectal carcinoma. Pretargeting using an huA33 immunoconjugate bearing a trans-cyclooctene-decorated dendritic scaffold (sshuA33-DEN-TCO) produced significantly higher tumoral activity concentrations at 120 h post-injection (23.0 ± 2.2 %ID/g) than those achieved with an analogous, dendrimer-lacking immunoconjugate (12.7 ± 2.6 %ID/g). However, pretargeting using sshuA33-DEN-TCO also resulted in increased activity concentrations in the blood at the same time point (1.9 ± 0.4 %ID/g) compared to the dendrimer-lacking construct (0.7 ± 0.2 %ID/g), thereby curtailing improvements to the tumor-to-blood therapeutic ratio of the system. In order to circumvent this issue, a tetrazine-labeled, dextran-based masking agent (Tz-DP) was injected prior to the radioligand to prevent the ligation between [177Lu]Lu-DOTA-PEG7-Tz and circulating immunoconjugate. This approach dramatically decreased the absorbed dose to the blood but also attenuated the absorbed dose to the tumor and increased the absorbed dose to the lungs. Ultimately, these data suggest that dendritic scaffolds and masking agents could be used to improve the dosimetry of PRIT, but the combination of these technologies will require extensive optimization.

Abstract A110: Efficacy of anti-PD1 immune checkpoint blockade involves the cooperative interaction of myeloid and lymphoid subpopulations in the tumor microenvironment

Abstract Suppression of the immune system by solid malignancies has proven to be a driving force of tumor development and an effective target for therapeutic intervention. The suppression of cytolytic T-cells by inhibitory receptors like PD1 can be blocked by antagonistic antibodies, reinvigorating suppressed antitumor responses. Nonetheless, only a minority of patients show clinical benefit. It is becoming clear the efficacy of checkpoint blockade relies on many factors, including pretreatment conditions, collaboration between innate and adaptive immune cells and immune-architecture of the tumor microenvironment (TME). To investigate this, we studied the immune profiles of the PD1-unresponsive murine B16 melanoma and PD1-responsive MC38 colorectal carcinoma models, systemically and in the TME before and during treatment. By using high-dimensional flow cytometry and unsupervised clustering analyses based on immune checkpoints, we show comparable early establishment of heterogeneity of tumor-infiltrating CD8+ and CD4+ T-cells. However, PD1-responsive MC38 tumor shows correlations in abundance between specific CD8+ T-cell, NK cells and myeloid subsets before checkpoint blockade treatment, while the PD1-unresponsive B16 tumors do not show lymphoid-myeloid codependences. Interestingly, the abundance of monocyte-derived dendritic cells did not increase upon anti-PD1 treatment but instead showed abundance correlation with PD1 CD4+ and CD8+ T-cells, suggesting a putative interaction. The unresponsive B16 tumors showed increased correlation of cDC1 and cDC2 with Foxp3+ regulatory T-cells. We have visualized these putative interactions within the myeloid and lymphoid population in the changing immune-architecture of the TME, using multiplex fluorescence and confocal microscopy. We show heterogeneity and interactive hotspots during immune checkpoint blockade and postulate that successful anti-PD1 treatment requires the location-dependent cooperation of specific myeloid and lymphoid subsets in the TME. Citation Format: Sjoerd Schetters, Yvette Van Kooyk. Efficacy of anti-PD1 immune checkpoint blockade involves the cooperative interaction of myeloid and lymphoid subpopulations in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A110.

Abstract B178: Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy

Abstract Background: The first cancer immunotherapy was developed in the 1890s by Dr. William Coley, who observed tumor regressions after injecting cancer patients with a heat-killed mixture of Gram-positive and negative pathogenic bacteria. Dr. Coley determined that the Gram-negative bacteria were the principal contributor to antitumor activity and that the product would likely be most effective when administered intravenously (i.v.), but was too toxic in this setting, limiting use to intramuscular and intratumoral administration. Coley’s toxins, as it was known, was credited with curing hundreds of late-stage cancer patients over 70 years. However, lack of knowledge regarding mechanism of action made it difficult to optimize or standardize, producing high variability. The use of multiple, local administration routes also likely contributed to variability in response, leading the FDA to refuse to grandfather in the product in 1963. We now know the mechanism of action of Coley’s toxins and the source of the i.v. toxicity. Gram-negative bacteria contain immune system danger signals, including multiple TLR agonists (activating TLRs 2, 4, 5 and 9), which directly and indirectly, via induction of cytokine and chemokine secretion, participate in the activation of most of the cellular mediators of innate and adaptive immune responses. Lipopolysaccharide (LPS), which activates TLR4, has been identified as a major contributor to both the antitumor activity and i.v. toxicity of Gram-negative bacteria. Decoy’s hypothesis is that significant reduction without complete elimination of LPS activity, in conjunction with killing and stabilization of nonpathogenic, Gram-negative bacteria, may produce a multiple TLR product that can safely and effectively induce antitumor immune responses via i.v. administration. Methods and Results: Nonpathogenic, Gram-negative E. coli were treated with polymyxin B and glutaraldehyde under conditions to kill and stabilize the cells, producing >90% reduction in LPS endotoxin activity and pyrogenicity. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate (LAL) and in vivo rabbit assays. Bacterial integrity was assessed by electron and light microscopy. Antitumor activity was determined using standard syngeneic and xenograft tumor models. Decoy-treated bacteria exhibited a 3-fold reduction in acute in vivo toxicity relative to untreated bacteria. Surprisingly, induction of antitumor cytokine secretion by murine and human peripheral blood mononuclear cells (PBMCs) was not compromised, relative to untreated bacteria. Treatment with Decoy bacteria (i.v.) produced significant single-agent antitumor activity against orthotopic murine colorectal carcinoma and metastatic murine pancreatic carcinoma. Synergistic combination activity, including eradication of established tumors, with a therapeutic index of up to 10-fold, was observed in combination with IL-2 or low-dose cyclophosphamide (LDC) in murine colorectal carcinoma models, with LDC in a subcutaneous (s.c.), murine non-Hodgkin’s lymphoma (NHL) model and with LDC plus rituximab in a s.c., human NHL model. Synergistic antitumor activity was also observed in combination with a low-dose, nonsteroidal anti-inflammatory drug (NSAID) in a metastatic, murine pancreatic carcinoma model. In addition, tumor eradications were observed in combination with NSAID and were enhanced by addition of anti-PD-1 therapy in a s.c., murine hepatocellular carcinoma model. Optimal (80-100%) tumor eradication was shown to be mediated by natural killer (NK), CD4+ and CD8+ T-cells. Immunologic memory (80-100% and partial), determined by rejection of subsequent tumor challenge, was demonstrated in both immune competent and innate only settings, respectively. Conclusion: We have developed an i.v. administered multiple TLR agonist, based on Coley’s toxins, that can be combined with a wide variety of existing agents to induce curative, antitumor immune responses. Citation Format: Michael J. Newman. Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B178.

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction.

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

ALPPS procedure in an experimental colorectal liver metastases model: hero or villain?

Background: Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been raised as a potential therapy for bilobar metastatic liver disease, although the potential tumor progression (TP) that this technique may induce is still not well established. Methods: The aim of this report was to study tumor hepatic progression induced by the first step of ALPPS in a WAG/Rij rat syngenic model of metastatic colorectal carcinoma by subcapsular inoculation of CC531 cell line. We analyzed the effect of hypoxia and immune response induced by ALPPS on liver metastases. Results: ALLPS seems to induce not only tumor progression, but also metastases independently of the site of inoculation. Immunohistochemical analisys revealed that ALPPS induced expression of hepatic vasculogenic factors and a dramatic increase of intrahepatic macrophages in comparison with non-operated groups. Interestingly, hepatic macrophages strongly expressed COX-2, while tumor-infiltrating macrophages expressed mainly arginase-1. ALPPS also induced a decrease of tumor-infiltrating lymphocytes and increase of intrahepatic T-lymphocytes. Conclusion: ALPPS technique may induce hypoxic environment which enhance hepatic HIF1alpha and VEGF expression. Additionally, the regenerative stimulus seems to be driven by a pro-inflammatory environment, in which M1 intrahepatic macrophages, which expressed COX-2 may develop a key role. These facts may be related with the tumor progression observed in inoculated and operated animals.

Biological evaluations of newly-designed Pt(II) and Pd(II) complexes using spectroscopic and molecular docking approaches

To perform biological evaluations of newly-designed Pt(II) and Pd(II) complexes, the present study was conducted with targeted protein human serum albumin (HSA) and HCT116 cell line as model of human colorectal carcinoma. The binding of Pt(II) and Pd(II) complexes to HSA was analyzed using fluorescence spectroscopy and molecular docking. The thermal stability and alterations in the secondary structure of HSA in the presence of Pt(II) and Pd(II) complexes were investigated using the thermal denaturation method and circular dichroism (CD) spectroscopy. The cytotoxicity of the Pt(II) and Pd(II) complexes was studied against the HCT116 cell line using MTT assay. The binding analysis revealed that the fluorescence findings were well in agreement with docking results such that there is only one binding site for each complex on HSA. Binding constants of 8.7 × 103 M−1, 2.65 × 103 M−1, 0.3 × 103 M−1, and 4.4 × 103 M−1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 25 °C, respectively. Also, binding constants of 1.9 × 103 M−1, 15.17 × 103 M−1, 1.9 × 103 M−1, and 13.1 × 103 M−1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 37 °C, respectively. The results of CD and thermal denaturation showed that the molecular structure of HSA affected by interaction with Pt(II) and Pd(II) complexes is stable. Cytotoxicity studies represented the growth suppression effect of the Pt(II) and Pd(II) complexes toward the human colorectal carcinoma cell line. Therefore, the results suggest that the new designed Pt(II) and Pd(II) complexes are well promising candidates for use in cancer treatment, particularly for human colorectal cancer.Communicated by Ramaswamy H. Sarma

Metabolic biomarkers of response to the AKT inhibitor MK-2206 in pre-clinical models of human colorectal and prostate carcinoma

BackgroundAKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition.MethodsCellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS.ResultsIn vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model.ConclusionThis MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.

Antisense Oligonucleotides against miR-21 Inhibit the Growth and Metastasis of Colorectal Carcinoma via the DUSP8 Pathway

Accumulating research has documented that microRNA-21 (miR-21) plays an important role in the development of human colorectal carcinoma (CRC). Our recent work also showed that antisense oligonucleotides (ASOs) against miR-21 can impair the growth of CRC cells in vitro. However, the potential role of miR-21 in gene therapy against CRC remains to be fully elucidated. Here, we further observed the effect of ASOs against miR-21 on the growth and metastasis of CRC in vivo using a xenograft model of human CRC. We found that ASOs could effectively inhibit the growth and metastasis of CRC in vivo, accompanied by downregulated expression of miR-21 and reduced transduction of the AKT and ERK pathway. Mechanically, global gene expression analysis showed that the expression of DUSP8, a novel target of miR-21, was upregulated in tumor mass. Furthermore, overexpression of DUSP8 could remarkably suppress the proliferation and migration of CRC cells in vitro. Finally, downregulation of DUSP8 could abrogate the effects of ASOs against miR-21 on the proliferation and migration of CRC cells, as well as altered transduction of the AKT and ERK signaling pathway. Together, these data suggest that ASOs against miRNAs are an attractive and potential therapeutic for the treatment of human CRC and warrant further development.

Abstract 2196: Noninvasive description of natural tumor evolution

Abstract Background: Our goal is to investigate how quantitative parameters of Contrast Enhanced Ultrasound (CEUS) and Shear Wave Elastography (SWE) change during tumor growth and link them with morphological and histological indexes. Methods: An ectopic model of murine colorectal carcinoma (CT26) was implanted in 46 mice (BALB/c ) at Day 0. On 5 subsequent days, data were acquired from subsets of these mice (D7, n=8 ; D10, n=9 ; D14, n=10 ; D16, n=9, D17, n=10) by scanning each tumor along its major, longitudinal axis in vivo to obtain conventional B-mode, SWE (SSI, Aixplorer, SL15-4 probe) and dynamic CEUS (Sequoia 512, 7-14 MHz probe, cadence contrast pulse sequencing) data. The ellipsoidal volume of each tumor was estimated from measurements of the width and thickness of the largest plane along the longitudinal and transversal plane using B-mode display on the Aixplorer device. Quantitative maps of tissue elasticity distribution in the tumor were obtained from SWE data. Average values and standard deviation of SWE data were calculated within regions selected to outline the tumor cross section on B-mode images. CEUS was performed after a syringe-pump-controlled bolus injection in the caudal vein of 40 μL of SonoVue (Bracco Suisse, Geneva, Switzerland) in a weight-adjusted volume of 0.5 mL/kg. A lognormal model was fit to the time intensity curve inside the tumor to estimate parameters related to microvascular volume and flow. All tumors imaged on a given day were excised and marked to conserve orientation and approximate position relative to the US imaging plane. Tumors were frozen with liquid nitrogen in an Optimal Cutting Temperature compound cube and stored at -80°C. Results: Significance of correlations were evaluated using the non-parametric Spearman correlation coefficient (RS). Average SWE parameters were not significantly correlated with the CEUS parameters (p > 0.03). Standard deviation of SWE in the tumor was well correlated with tumor volume (RS = 0.65 [p<6x10^-7]). For CEUS only the Time To Peak and Latency Time correlated with tumor volume (RS = 0.62 [p<4x10^-6] and RS = -0.72 [p<2x10^-8], respectively). Correlation with histological assessment of tumor microstructure is still in the process of analysis. Conclusions: These preliminary results probe the relationship between tumor volume, functional-flow and elastic properties in tumors. Both CEUS and SWE parameters were found that correlate with tumor growth and thus, appear to be sensitive to changes occurring during tumor development. Histological analysis of tumor microstructure is underway to more fully probe this point. Lack of correlation between SWE and CEUS parameters suggests that they provide complementary information. Better understanding of the sensitivity of these non invasive parameters to tumor growth changes will contribute to their eventual integration in procedures of tumor response criteria in clinical practice. Citation Format: Jerome Griffon, Delphine Le Guillou-Buffello, Oumeima Laifa, Lori Bridal, Michele Lamuraglia. Noninvasive description of natural tumor evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2196.

Abstract 3851: Novel small-molecule inhibitor of PD1/PDL1 pathway demonstrated single agent and drug combo effectiveness in cancer immunotherapy

Abstract With 5 antibodies approved for cancer treatments, PD-1/PD-L1 pathway has been the most successful target in cancer Immuno-therapy. Small molecules that inhibit PD-1/PD-L1 interaction are also attractive because their better tissue penetration may lead to stronger and broader anti-tumor efficacy in addition to more convenient dosing regimen. A series of such small molecule inhibitors have been developed by Maxinovel. Among them, Max 10043 and its 2nd generation compound MAX-10129 potently inhibited PD-1/PD-L1 interaction, reversed PD-L1 suppression of anti-CD3 mediated activation of human T cells, and was well tolerated in animal studies. MAX-10043 also demonstrated anti-tumor efficacy in CT26 murine colorectal carcinoma model. MAX-10129, with improved oral bioavailability, displayed significant inhibition of tumor progression with dose dependency in murine colorectal carcinoma MC-38 model. MAX-10129 also demonstrated synergistic anti-tumor efficacy in different combinations with an anti-CTLA4 antibody, an IDO inhibitor Epacadostat, a COX-2 inhibitor Celebrex and a chemo drug Cisplatin, respectively. The p value of the combo treatment group relative to the vehicle group is less than 0.01 for the Epacadostat combo, the Celebrex Combo and the Cisplatin combo while it is less than 0.05 for the anti-CTLA4 combo. More studies are on-going to illustrate mechanism of action as well as the possibility of all oral triple and quadruple combinations . These data suggest the MAX-10129, a small molecule inhibitor of PD-1/PDL-1 interaction, may represent a novel, orally administrated, safe and effective immunotherapy agent. Citation Format: Yuguang Wang, He Zhou, Nong Zhang, Feilan Wang, Qiang Zhao, Tianzhi Wu, Haiyan Zhu, Yuzhi Liu. Novel small-molecule inhibitor of PD1/PDL1 pathway demonstrated single agent and drug combo effectiveness in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3851.

PO-030 Functional analysis of mastl mutations in cancer

IntroductionMastl, also known as Greatwall, is a protein kinase essential for proper chromosome condensation and progression through mitosis and meiosis. It belongs to the AGC kinase family and, particularly, presents a non-conserved insertion of 550 aa at the corresponding T-activation loop site in the C-lobe (usually 20–30 aa). This non-conserved middle region (NCMR) is not considered to have an essential role for Mastl activity but its function in unknown. Mastl is involved in the inhibition of protein phosphatase 2A (PP2A)-B55 complexes to maintain the mitotic state. By using a conditional knockout in mouse generated in our lab, it was shown that mammalian Mastl is essential for mouse embryonic development and cell cycle progression. Mastl was initially found in humans as a gene mutated in thrombocytopenia and preliminary data suggests its overexpression in tumours. However, very little is known about this protein in human disease.Material and methodsGenomic data from repositories of cancer somatic mutations include MASTL NCMR indel mutations, leading to the generation of a truncated protein. Exome sequencing studies in Mastl in gastric cancers show that mutant tumours present microsatellite instability (MSI). We have studied the prevalence of these mutations by sequencing MASTL in a subset of samples from colon and stomach patients.To evaluate the therapeutic relevance of this kinase, functional assays have been performed, as complementation studies and kinase assays.To mimic the cancer mutations we have generated a new mouse model using CRISPR/Cas9 technology. We are currently performing several assays such as focus assays, scratch assays, soft agar and colony formation on cells derived from this model. In addition, we are using a chemical-induced colorectal carcinoma model to study the role of these mutant kinases in cancer.Results and discussionsA heterozygous exonic indel mutation has been found in an MSI +CRC from the 21 patient samples sequenced.Functional assays with the mutant enzyme resulted in a partial rescue in DNA segregation observed in Mastl-null cells.Mastl mutant forms resulted in embryonic lethality in homozygosis. Therefore, our carcinogenesis models are performed in heterozygous mice, thus mimicking cancer mutations.ConclusionMastl indel mutations in the NCMR region lead to the expression of truncated shorter forms. These Mastl mutated forms are not able to fully accomplish the role of Mastl in mitosis. Mutant heterozygous mice, mimicking MASTL cancer mutations, are viable and fertile.

Role of Kupffer cells in the progression of CRC liver metastases after the first stage of ALPPS

Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been suggested as a potential therapy for extensive bilobar liver tumors, although in some circumstances this technique may induce tumor progression, a fact still not well studied. Our aim was to study tumor hepatic progression induced by the first step of ALPPS in a WAG/Rij rat syngenic model of metastatic colorectal carcinoma by subcapsular CC531 cell line inoculation. ALPPS induced: tumor progression on deportalized lobe and metastases; expression of hepatic vasculogenic factors (HIF1-α and VEGF); and a dramatic increase of Kupffer cells (KCs) and tumor-associated macrophages (TAMs). Interestingly, KCs expressed COX-2 (M1 polarization), while TAMs expressed mainly arginase-1 (M2 polarization). ALPPS also induced a decrease of tumor-infiltrating lymphocytes and an increase of intrahepatic T lymphocytes. Thus, ALPPS technique seems to induce a hypoxic environment, which enhances hepatic HIF1-α and VEGF expression and may promote KCs and TAMs polarization. Consequently, the regenerative stimulus seems to be driven by a pro-inflammatory and hypoxic environment, in which M1 intrahepatic macrophages expressing COX-2 and T-Lymphocytes play a key role, facts which may be related with the tumor progression observed.

MicroRNA-451a regulates colorectal cancer proliferation in response to radiation

BackgroundColorectal cancer (CRC) is a leading cause of cancer-related death. The biologic response of CRC to standard of care adjuvant therapies such as chemotherapy and radiation are poorly understood. MicroRNAs (miRs) have been shown to affect CRC progression and metastasis. Therefore, we hypothesized that specific miRs modulate CRC response to chemoradiation.MethodsIn this study, we used miR expression profiling and discovered a set of microRNAs upregulated rapidly in response to either a single 2 Gy dose fraction or a 10 Gy dose of γ-radiation in mouse colorectal carcinoma models. We used gain and loss-of-function studies in 2D and 3Dcell proliferation assays and colony formation assays to understand the role of the top miR candidate from our profiling. We used Student’s T-tests for simple comparisons and two-factor ANOVA for evaluating significance.ResultsThe most upregulated candidate at early time points in our signature, miR-451a inhibited tumor cell proliferation and attenuated surviving fraction in longer-term cultures. Conversely, inhibition of miR-451a increased proliferation, tumorsphere formation, and surviving fraction of tumor cells. Using a bioinformatics approach, we identified four genes, CAB39, EMSY, MEX3C, and EREG, as targets of miR-451a. Transfection of miR-451a decreased both mRNA and protein levels of these targets. Importantly, we found miR-451a expression was high and CAB39, EMSY levels were low in a small subset of rectal cancer patients who had a partial response to chemoradiation when compared to patients that had no response. Finally, analysis of a TCGA colorectal cancer dataset revealed that CAB39 and EMSY are upregulated at the protein level in a significant number of CRC patients. Higher levels of CAB39 and EMSY correlated with poorer overall survival.ConclusionsTaken together, our data indicates miR-451a is induced by radiation and may influence colorectal carcinoma proliferation via CAB39 and EMSY pathways.

A Tricin Derivative from Deschampsia antarctica Desv. Inhibits Colorectal Carcinoma Growth and Liver Metastasis through the Induction of a Specific Immune Response

In colorectal carcinoma patients, distant metastatic disease is present at initial diagnosis in nearly 25% of them. The majority of patients with metastatic colorectal carcinoma have incurable disease; therefore, new therapies are needed. Agents derived from medicinal plants have already demonstrated therapeutic activities in human cancer cells. Antartina is an antitumor agent isolated from Deschampsia antarctica Desv. This study aimed to evaluate the antitumor properties of Antartina in colorectal carcinoma models. We used human and murine colorectal carcinoma cell lines for investigating proliferation, apoptosis, and cell-cycle effects of Antartina therapy in vitro Avatar and immunocompetent colorectal carcinoma animal models were applied for evaluating the effects of Antartina in vivo Immune response against colorectal carcinoma model was investigated using CTL assay, analyzing dendritic cell activation and intratumor T-cell subpopulation, and by tumor rechallenge experiments. Antartina inhibits in vitro human colorectal carcinoma cell proliferation; however, in vivo experiments in Avatar colorectal carcinoma model Antartina display a limited antitumor effect. In an immunocompetent colorectal carcinoma mice model, Antartina potently inhibited tumor growth and liver metastases, leading to complete tumor regressions in >30% of mice and increased animal survival. In addition, Antartina induced a potent specific cytotoxic T-cell response against colorectal carcinoma and a long-lasting antitumor immunity. Interestingly, Antartina increased tumor immunogenicity and stimulated dendritic cell activation. No toxic effects were observed at the doses employed. Our findings showed that Antartina has the ability to induce antitumor immunity against colorectal carcinoma and can be used to develop new tools for the treatment of colorectal carcinoma. Mol Cancer Ther; 17(5); 966-76. ©2018 AACR.

Investigating Low-Velocity Fluid Flow in Tumors with Convection-MRI.

Several distinct fluid flow phenomena occur in solid tumors, including intravascular blood flow and interstitial convection. Interstitial fluid pressure is often raised in solid tumors, which can limit drug delivery. To probe low-velocity flow in tumors resulting from raised interstitial fluid pressure, we developed a novel MRI technique named convection-MRI, which uses a phase-contrast acquisition with a dual-inversion vascular nulling preparation to separate intra- and extravascular flow. Here, we report the results of experiments in flow phantoms, numerical simulations, and tumor xenograft models to investigate the technical feasibility of convection-MRI. We observed a significant correlation between estimates of effective fluid pressure from convection-MRI with gold-standard, invasive measurements of interstitial fluid pressure in mouse models of human colorectal carcinoma. Our results show how convection-MRI can provide insights into the growth and responsiveness to vascular-targeting therapy in colorectal cancers.Significance: A noninvasive method for measuring low-velocity fluid flow caused by raised fluid pressure can be used to assess changes caused by therapy. Cancer Res; 78(7); 1859-72. ©2018 AACR.

MUC1- and Survivin-based DNA Vaccine Combining Immunoadjuvants CpG and interleukin-2 in a Bicistronic Expression Plasmid Generates Specific Immune Responses and Antitumour Effects in a Murine Colorectal Carcinoma Model.

DNA vaccination is a promising cancer treatment due to its safety, but poor immunogenicity limits its application. However, immunoadjuvants, heterogeneous prime-boost strategies and combination with conventional treatments can be used to improve the antitumour immune effects. A CpG motif and interleukin-2 (IL-2) cytokine are often used as adjuvants. In this study, a DNA vaccine containing a CpG motif was constructed to evaluate its adjuvant effect. The results show that the cytotoxicity of the DNA vaccine was increased fivefold, and survival lifetime was prolonged twofold by the CpG motif adjuvant. To simplify the industrial production process, a bicistronic plasmid was constructed to carry the fusion genes of survivin/MUC1 (MS) and IL-2 and with a CpG motif in its backbone. The results showed that the antitumour effect of the bicistronic vaccine was the same as that of the two vaccine co-injected regime. Furthermore, the vaccine could suppress metastatic tumour foci by 69.1% in colorectal carcinoma-bearing mice. Moreover, the vaccine induced survivin- and MUC1-specific immune responses in splenocytes and induced the immune promoting factor CCL-19 and GM-CSF upregulated, while metastatic-associated factor MMP-9 and immunosuppressing factor PD-L1 downregulated in tumour tissue. When combining the vaccine with the chemotherapy drug oxaliplatin, the survival was prolonged by about 2.5-fold. In conclusion, the DNA vaccine containing a CpG motif in bicistronic form showed good effects on colorectal cancer by inhibiting both tumour growth and metastasis, and combination with oxaliplatin could improve its antitumour effects.

Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention.

Chemoprevention-the use of medication to prevent cancer-can be augmented by the consumption of produce enriched with natural metabolites. However, chemopreventive metabolites are typically inactive and have low bioavailability and poor host absorption. Here, we show that engineered commensal microbes can prevent carcinogenesis and promote the regression of colorectal cancer through a cruciferous vegetable diet. The engineered commensal Escherichia coli bound specifically to the heparan sulphate proteoglycan on colorectal cancer cells and secreted the enzyme myrosinase to transform host-ingested glucosinolates-natural components of cruciferous vegetables-to sulphoraphane, an organic small molecule with known anticancer activity. The engineered microbes coupled with glucosinolates resulted in >95% proliferation inhibition of murine, human and colorectal adenocarcinoma cell lines in vitro. We also show that murine models of colorectal carcinoma fed with the engineered microbes and the cruciferous vegetable diet displayed significant tumour regression and reduced tumour occurrence.