Tumor Xenografts In Immunodeficient Mice Research Articles

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

Synergistic inhibitory effect of α-humulene and sclareol on human pancreatic cancer cells

• α-Humulene and sclareol showed synergistic inhibitory effect on human pancreatic cancer cells. • The combination strongly decreased the levels of NF-κB, phosphor Akt (pAkt) and Bcl-2, and increased the level of Bax. • The in vivo study showed that HUM and SCL in combination suppressed the growth of Panc-1 xenograft tumors in immunodeficient mice. Both α-Humulene (HUM) and sclareol (SCL) are naturally occurred compounds and have been shown to exert anti-inflammatory and anticancer activities. The effects of these two compounds alone or in combination on pancreatic cancer cells have not been reported. In the present study, we investigated the effects of HUM and SCL on human pancreatic cancer cells cultured in vitro and grown as xenograft tumors in vivo. In the in vitro studies, we found that HUM and SCL in combination synergistically inhibited the growth of pancreatic cancer cells. The combination strongly stimulated apoptosis, and suppressed migration, invasion and sphere formation in pancreatic cells. Studies on the mechanism of action showed that the combination strongly decreased the levels of NF-κB, phosphor Akt (pAkt) and Bcl-2, and increased the level of Bax. Knockdown of NF-κB and Akt by small interfering RNA (siRNA) abrogated the inhibitory effect of HUM and SCL on pancreatic cancer cells. The in vivo study showed that HUM and SCL in combination suppressed the growth of Panc-1 xenograft tumors in immunodeficient mice. Results of the present study indicate that HUM and SCL in combination may represent an effective approach for inhibiting the growth of pancreatic cancer.

SRT1720 induces SIRT1-independent cell death in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops after a long period of human T-cell leukemia virus (HTLV)-1 infection and is associated with host aging in addition to genetic abnormalities in HTLV-1 infected cells. SIRT1 is a histone deacetylase involved in cell cycle and apoptosis. We previously showed the high expression of SIRT1 protein in peripheral blood mononuclear cells from patients with ATL. There have been many reports that SIRT1 inhibitors show tumor-suppressive effects. On the other hand, SIRT1 activator SRT1720 induces the cell death of multiple myeloma and breast cancer cells. However, the effect of SRT1720 on ATL is unknown. This study aimed to evaluate the effect of SRT1720 on cell death in leukemic cell lines in vitro and freshly isolated ATL cells ex vivo and in an ATL in vivo mouse model. SRT1720 reduced cell viability in vitro and ex vivo. Additionally, SRT1720 increased the number of apoptotic cells, as shown by annexin V positive cells, cleaved poly (ADP-ribose) polymerase 1, cleaved caspase-3, and fragmented DNA. SRT1720 also induced mitochondrial outer membrane permeabilization with the generation of mitochondrial reactive oxygen species and autophagy. However, SIRT1 knockdown did not attenuate SRT1720-induced cell death in leukemic cell lines. Finally, SRT1720 treatment decreased the growth of human ATL tumor xenografts in immunodeficient mice. Our study shows that while SRT1720 does not target SIRT1, it induces cell death in ATL cells via apoptosis and autophagy and has antitumor activity.

Selective Targeting of Class I Histone Deacetylases in a Model of Human Osteosarcoma.

Simple SummaryOsteosarcoma is the predominant form of primary bone cancer and outcomes for patients with metastatic disease have not improved for several decades. Recent genomic and pharmacological studies have implicated dysregulated histone deacetylases as druggable targets to treat several types of cancers, including osteosarcoma. This study aimed to assess the inhibitory effects of 4SC-202, a next-generation inhibitor for class I histone deacetylases, on human osteosarcoma cell growth in vitro and in vivo. We demonstrated that the anti-tumor effect of 4SC-202 involves combined cell-cycle arrest and apoptosis, as well as a reduction in cell invasion and migration. Moreover, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Additionally, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients.Dysregulation of histone deacetylases (HDACs) is associated with the pathogenesis of human osteosarcoma, which may present an epigenetic vulnerability as well as a therapeutic target. Domatinostat (4SC-202) is a next-generation class I HDAC inhibitor that is currently being used in clinical research for certain cancers, but its impact on human osteosarcoma has yet to be explored. In this study, we report that 4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By analyzing cell function in vitro, we show that the anti-tumor effect of 4SC-202 involves the combined induction of cell-cycle arrest at the G2/M phase and apoptotic program, as well as a reduction in cell invasion and migration capabilities. We also found that 4SC-202 has little capacity to promote osteogenic differentiation. Remarkably, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Moreover, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. We further reveal key targets regulated by 4SC-202 that contribute to tumor cell growth and survival, and canonical signaling pathways associated with progression and metastasis of osteosarcoma. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients with osteosarcoma and provide molecular insights into the mechanism of action of class I HDAC inhibitors.

Abstract 305: ELU001, a targeted C'Dot drug conjugate (CDC) for the treatment of folate receptor alpha (FRα) overexpressing cancers

Abstract CDCs are novel ultra-small (6-7 nm) nanoparticle drug conjugates that have been demonstrated to be capable of faster tumor targeting and deeper tumor penetration than antibody drug conjugates in animal models. CDCs are capable of targeting tumors in the brain and pancreas that are difficult to access, while exhibiting limited exposure to normal tissues due their efficient renal elimination. CDCs are composed of a silica core, in which Cy5, a far red dye is covalently encapsulated. The silica core is covalently coated with a layer of polyethylene glycol which is then functionalized with targeting moieties and payloads. ELU001 is a CDC functionalized with ~20 molecules of the topoisomerase-1 inhibitor exatecan linked via a proteolytic cleavable linker as a payload and ~15 folic acids to provide targeting to FRα overexpressing cancers. ELU001 is rapidly internalized into FRα expressing cells and is trafficked to the lysosome where the payload is released from the CDC. ELU001 exhibits potency in the low single digit nanomolar to sub-nanomolar range against cancer cells that express 3+ (KB, IGROV-1) and 2+ (SK-OV-3, HCC827 and OVCAR-3) levels of FRα after a 6-hr exposure in a 7-day viability study. In contrast, an anti-FRα ADC based ADC mirvetuximab soravtansine exhibits lower potency (>100 nM IC50) against SK-OV-3 and HCC827 cells and 40 nM IC50 against OVCAR-3 cells. ELU001 exhibits potent efficacy against established s.c. KB human cervical tumor xenografts in immunodeficient mice with significantly better efficacy and safety than free exatecan payload. It is also effective in treating established SK-OV-3 tumors with lower (2+) FRα expression, a setting where the ADC is again less effective. IND-enabling nonclinical studies are currently underway to prepare for initiation of a first-in-human phase 1 clinical trial in subjects with FRα overexpressing cancers in the second half of 2021. Citation Format: Gregory Paul Adams, Kai Ma, Aranapakam Venkatesan, Feng Chen, Fei Wu, Melik Turker, Thomas Gardinier, Peiming Chen, Vaibhav Patel, Eliel Bayever, Paul Rudick, Geno Germano. ELU001, a targeted C'Dot drug conjugate (CDC) for the treatment of folate receptor alpha (FRα) overexpressing cancers [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 305.

Abstract 1710: A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo

Abstract Tumor-Specific Immuno-Gene (T-SIGn) cancer gene therapy vectors, which are based on the oncolytic adenovirus enadenotucirev, carry a gene cassette encoding combinations of human transgenes under control of the virus major late promoter (MLP) which restricts expression to tumor cells. These vectors can be dosed intravenously for delivery to tumors and selective expression of therapeutic proteins locally within the tumor microenvironment. Because these viruses only infect human tumor cells, in vivo studies are limited to human tumor xenografts in immunodeficient mice. Recently, we have optimized a 3D microfluidic system for evaluating response to immunotherapies using patient-derived organotypic tumor spheroids (PDOTS). PDOTS contain stromal as well as autologous myeloid and lymphoid cell populations and respond to PD-1 blockade in short-term ex vivo culture. As an alternative preclinical approach for evaluating activity and mechanistic underpinnings of T-SIGn viruses expressing human immunomodulatory transgenes, we have been developing the PDOTS ex vivo culture model. Here we report on the visualization of oncolytic virus uptake, replication in tumor cells and activation of T cells with a T-SIGn virus encoding an EpCAM-targeted bispecific T-cell activator (NG-611) within NSCLC PDOTS. Surgical NSCLC cases from St. Elizabeth's Medical Center, collected under an IRB approved protocol, were processed in PDOTS and assessed for EpCAM expression and number of T cells. Five different samples were treated with NG-611 (mixed with a GFP expressing vector) at concentrations ranging from 2x109 to 2x106 vp/mL final volume of culture. Virus infection was shown after 3 and 5 days of culture by dose-dependent expression of GFP reporter transgene driven by a CMV promoter, and virus replication in tumor cells was confirmed by using GFP expressed under the virus MLP promoter. Upregulation of IFNγ and Granzyme B in culture media indicated a strong activation of T-cells via expression of the bispecific T-cell activator. Cytotoxic T-cell activation also led to dose-dependent cell killing in NSCLC spheroids as shown by life/death analysis. Having established this proof of concept for functionality of T-SIGn viruses in PDOTS, ongoing studies are evaluating the immunomodulatory activities of a series of T-SIGn viruses expressing human IL-12 with or without other cytokine/chemokines. Citation Format: Moataz Noureddine, Katy West, Elizabeth Isaacson, Sylvia Alarcon, Christopher S. Lathan, John Wain, David A. Barbie, Cloud P. Paweletz, Elena Ivanova, Brian R. Champion. A T-SIGn cancer gene therapy virus expressing an EpCAM-targeted bispecific T-cell activator stimulates T-cells and induces tumor cell death in patient-derived organotypic non-small cell lung cancer (NSCLC) spheroids ex vivo [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 1710.

Alantolactone suppresses the metastatic phenotype and induces the apoptosis of glioblastoma cells by targeting LIMK kinase activity and activating the cofilin/G‑actin signaling cascade.

Glioblastoma (GBM) is the most common aggressive brain tumor and is associated with an extremely poor prognosis, as the current standard of care treatments have limited efficacy. Natural compounds have attracted increasing attention as potential anticancer drugs. Alantolactone (ATL) is a natural small molecule inhibitor, that has antitumor properties. In the present study, U87MG and U251 cells were treated ATL and changes in actin/G-actin/F-actin/cofilin pathway were detected in whole cells, in the cytoplasm and mitochondria by western blot analysis. Immunofluorescence and immunoprecipitation analysis identified changes in the expression levels of target proteins and interactions, respectively. A LIMK enzyme inhibitor was also applied to assess the effects of ATL on the migration and invasion of GBM cells. Flow cytometry was used to detect the levels of apoptosis of GBM cells. The expression of matrix metalloproteinase (MMP)-2/MMP-9, caspase-3/caspase-9/poly(ADP-ribose) polymerase (PARP)/cytochrome c, were determined by western blot analysis to assess the effects of targeting LIMK. The in vitro findings were verified in vivo by characterizing changes in the expression of cofilin/LIMK in xenograft tumors in immunodeficient mice. It was found that ATL activated cofilin through the targeted inhibition of LIMK enzyme activity and it thus upregulated the ratio of G/F actin, and inhibited GBM cell migration and invasion. Conversely, the activation of cofilin and G-actin could be co-transferred to the mitochondria to initiate the mitochondrial-cytochrome c pathway to induce apoptosis. On the whole, the findings of the present study further illustrate the molecular mechanisms through which ATL inhibits the metastatic phenotype of GBM cells and induces apoptosis. Given previous findings, it can be deduced that ATL can function through multiple pathways and has multiple targets in GBM models, highlighting its potential for use in clinical applications.

In vivo and in vitro cell-based model of lung adenocarcinoma from patients with pleural effusion.

Lung adenocarcinoma (LAC) is a common and aggressive form of lung cancer that is increasing in incidence among never smokers at a younger age. Current treatment of patients with LAC is insufficient and there is a need for identification of effective biomarkers and development of therapeutic targets. These demands require also improved models for in vivo and in vitro experimentation. In this study, we describe the establishment of two LAC cell lines, named LuCa-3 and LuCa-6. Both were derived from pleural effusion (PE) cells of LAC patients (L3 and L6) and readily propagated as tumor xenografts in immunodeficient mice. PE cells from the patient L6 exhibited also the capacity for in vitro growth and were cultured in two forms: (i) as a suspension growing cell population, labeled LuCa-6S, composed of non-clumping single cells; and (ii) as a monolayer-like culture, labeled LuCa-6A, exhibiting tight cell-to-cell and to culture surface adherence. Unique features of these two sublines and their cell clones are the capacity to convert from a non-clumping single-cell suspension into the adherent growth pattern and vice versa. Immunostaining of patients' tumor tissue xenografts and cultured subline cells displayed markers specific for the phenotype of human LAC. LuCa-6S and LuCa-6A cells did not reveal a noticeable disparity in quantitative growth characteristics. However, a number of differences were detected between these two cell populations manifested in detection or intensities of antigen expressions on the cell surface (CD133, SFTPC) and in the nucleus (TTF-1) including pluripotent (OCT-4, SOX-2, NANOG) genes in cancer stem-like cells (CSCs). Dissimilarities between these two sublines were also detected in N-glycan profiles and in the sensitivity to natural killer cells. Salient features of these subline cell populations are responsiveness to selective upregulation of the pluripotent genes in subsets of CSCs via conversion of their growth patterns and/or by using culture stem media with growth factors. The described in vivo/in vitro model enables broader experimental approaches in studies of lung adenocarcinoma.

Mild Whole-Body Hyperthermia-Induced Interstitial Fluid Pressure Reduction and Enhanced Nanoparticle Delivery to PC3 Tumors: In Vivo Studies and Micro-Computed Tomography Analyses

Abstract In this study, we performed in vivo experiments on mice to evaluate whether whole-body hyperthermia enhances nanoparticle delivery to PC3 (prostatic cancer) tumors. PC3 xenograft tumors in immunodeficient mice were used in this study. The mice in the experimental group were subjected to whole-body hyperthermia by maintaining their body temperatures at 39–40 °C for 1 h. Interstitial fluid pressures (IFPs) in tumors were measured before heating, immediately after, and at 2 and 24 h postheating in both the experimental group and in a control group (without heating). A total of 0.2 ml of a newly developed nanofluid containing gold nanoparticles (AuNPs) was delivered via the tail vein in both groups. The micro-computed tomography (microCT) scanned images of the resected tumors were analyzed to visualize the nanoparticle distribution in the tumors and to quantify the total amount of nanoparticles delivered to the tumors. Statistically significant IFP reductions of 45% right after heating, 47% 2 h after heating, and 52% 24 h after heating were observed in the experimental group. Analyses of microCT scans of the resected tumors illustrated that nanoparticles were more concentrated near the tumor periphery rather than at the tumor center. The 1-h whole-body hyperthermia treatment resulted in more nanoparticles present in the tumor central region than that in the control group. The mass index calculated from the microCT scans suggested overall 42% more nanoparticle delivery in the experimental group than that in the control group. We conclude that 1-h mild whole-body hyperthermia leads to sustained reduction in tumoral IFPs and significantly increases the total amount of targeted gold nanoparticle deposition in PC3 tumors. The present study suggests that mild whole-body hyperthermia is a promising approach for enhancing targeted drug delivery to tumors.

Inhibition of p62/SQSTM1 sensitizes small-cell lung cancer cells to cisplatin-induced cytotoxicity by targeting NEDD9 expression.

Drug resistance is the leading cause for rapid progression and relapse in small-cell lung cancer (SCLC) patients. Thus overcoming drug resistance still remains to be urgently resolved during SCLC treatment. Here, we found p62/SQSTM1 was enriched in SCLC spheroids, a subpopulation possessing cancer stem-like properties, which is responsible for cancer relapse and metastasis. Subsequent functional assays in vitro showed that short hairpin RNA (shRNA)-mediated p62 knockdown increased sensitivity of SCLC cell lines to cisplatin (DDP), whereas lentivirus-mediated p62 ectopic overexpression diminished DDP-induced cytotoxicity in both NCI-H446 and NCI-H1688 cell lines. Moreover, ectopic p62 overexpression promoted DDP resistance of NCI-H446 cells-derived tumor xenografts in immunodeficient mice in vivo, as indicated by accelerated tumor growth rate and reduced fluorescent activity of cleaved caspase-3. Gene expression profiling analysis revealed that p62 was positively correlated with neuronal precursor cell-expressed, developmentally downregulated gene 9 (NEDD9) expression level. Consistently, NEDD9 messenger RNA (mRNA) level was decreased upon p62 suppression by small interfering RNA (siRNA) and increased with p62 transient overexpression in SCLC cell lines, suggesting that p62 positively regulated NEDD9 mRNA. Depletion of NEDD9 by siRNA, to a large extent, reversed p62-overexpressed SCLC cells to DDP-induced cytotoxicity, implying NEDD9 might act as a downstream target which was in charge of p62-mediated DDP resistance. Taken together, our findings uncovered a previously unknown role of p62 in the regulation of SCLC drug resistance, assigning p62 as an attractive target for SCLC treatment.

Therapeutic Use of Vitamin C in Cancer: Physiological Considerations.

Since the early studies of William J. McCormick in the 1950s, vitamin C has been proposed as a candidate for the treatment of cancer. A number of reports have shown that pharmacological concentrations of vitamin C selectively kill cancer cells in vitro and decrease the growth rates of a number of human tumor xenografts in immunodeficient mice. However, up to the date there is still doubt regarding this possible therapeutic role of vitamin C in cancer, mainly because high dose administration in cancer patients has not showed a clear antitumor activity. These apparent controversial findings highlight the fact that we lack information on the interactions that occurs between cancer cells and vitamin C, and if these transformed cells can uptake, metabolize and compartmentalize vitamin C like normal human cells do. The role of SVCTs and GLUTs transporters, which uptake the reduced form and the oxidized form of vitamin C, respectively, has been recently highlighted in the context of cancer showing that the relationship between vitamin C and cancer might be more complex than previously thought. In this review, we analyze the state of art of the effect of vitamin C on cancer cells in vitro and in vivo, and relate it to the capacity of cancer cells in acquiring, metabolize and compartmentalize this nutrient, with its implications on the potential therapeutic role of vitamin C in cancer.

High expression of NAMPT in adult T-cell leukemia/lymphoma and anti-tumor activity of a NAMPT inhibitor

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes induced by human T-cell leukemia virus-1 and has a poor outcome. New molecular targets for the prevention and treatment of ATL are needed urgently. We previously reported high expression of Sirtuin 1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, in primary acute-type ATL cells. NAD+ biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) modulates Sirtuin 1 activity. Here, we examined the expression and effects of inhibiting NAMPT, a rate-limiting enzyme in NAD+ biosynthesis, in ATL cells. We found that peripheral blood mononuclear cells from patients with acute-type ATL expressed significantly higher levels of NAMPT protein than cells from healthy subjects. FK866, a NAMPT inhibitor, induced apoptosis of freshly isolated ATL cells ex vivo and HTLV-1-infected T-cell lines in vitro, which was accompanied by activation of caspases, DNA fragmentation, and disruption of mitochondrial transmembrane potential. However, a pan-caspase inhibitor failed to prevent this FK866-induced cell death, while FK866 increased the caspase-independent cell death mediator endonuclease G. Intriguingly, FK866 also activated autophagy, as demonstrated by increases in protein levels of autophagosome marker LC3-II. Thus, FK866 simultaneously activated apoptosis and autophagy. Finally, FK866 treatment markedly decreased the growth of human ATL tumor xenografts in immunodeficient mice. We showed that NAMPT is highly expressed in primary ATL cells ex vivo, and that FK866 induces autophagy and caspase-dependent and -independent cell death pathways in vitro and has an anti-tumor activity in vivo. These results suggest a novel therapeutic strategy for patients with this fatal disease.

41P - LCSCAF1 maintains cancer stem-like traits by stabilizing c-Myc protein and promotes metastasis and recurrence in lung cancer

BackgroundAberrantly overexpressed lung cancer stem cell associated factor-X (LCSCAFX) has been implicated in non-small cell lung cancer (NSCLC) development. MethodsTo explore the effect of LCSCAFXon the lung cancer stem-like traitsin vitro and in vivo. ResultsWe reported that enforced expression of LCSCAFX promoted NSCLC cells invasion and metastasis, drug resistance and recurrencein vitro and in vivo, whereas depletion of LCSCAFX suppressed metastasis and relapse in NSCLC. Moreover, we found that LCSCAFX expression was elevated in lung cancer stem cells (LCSCs). Indeed, knockdown of LCSCAFX impaired lung cancer stem-like traits. Conversely, ectopic overexpression of LCSCAFX enhanced the self-renewal ability of lung cancer cells in vitro. In addition, elevated LCSCAFX robustly enhanced tumor initiating frequencies, as well as growth rates of NSCLC cells derived tumor xenografts in immunodeficient mice. Furthermore, mechanistic investigations established that LCSCAFX stabilized c-Myc protein by suppressing its ubiquitylation and proteasomal degradation, which is required for lung cancer stem-like properties maintenance. Consistently, immunohistochemical analysis of clinical lung tumor tissues showed that LCSCAFX was elevated in NSCLC tissues and high LCSCAFX expression serve as an independent prognostic marker in patients with NSCLC. Importantly, genetic depletion of LCSCAFX combined chemotherapy dramatically inhibited NSCLC progression in patient-derived xenograft (PDX) model. ConclusionsThese findings demonstrated a previously unappreciated role of LCSCAFX in the regulation of LCSCs, assigning LCSCAFX as a promising therapeutic target for lung cancer treatments. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Cholesterol Depletion by TASIN-1 Induces Apoptotic Cell Death through the ER Stress/ROS/JNK Signaling in Colon Cancer Cells.

Truncated APC selective inhibitor-1 (TASIN-1) is a recently identified small molecule that selectively kills colorectal cancer cells that express truncated adenomatous polyposis coli (APC) by reducing cellular cholesterol levels. However, the downstream mechanism responsible for its cytotoxicity is not well understood. In this study, we show that TASIN-1 leads to apoptotic cell death via inducing ER stress-dependent JNK activation in human truncated APC colon cancer cells, accompanied by production of reactive oxygen species (ROS). In addition, TASIN-1 inhibits AKT activity through a cholesterol-dependent manner. Human colon tumor xenografts in immunodeficient mice also show the same TASIN-1 induced molecular mechanisms of tumor cell death as observed in vitro Taken together, cholesterol depletion by TASIN-1 treatment induces apoptotic cell death through activating ER stress/ROS/JNK axis and inhibiting AKT pro-survival signaling in colon cancer cells with truncated APC both in vitro and in vivoMol Cancer Ther; 17(5); 943-51. ©2018 AACR.

3D cellular spheroids as tools for understanding carboxylated quantum dot behavior in tumors

BackgroundMonolayer cell cultures have been considered the most suitable technique for in vivo cellular experiments. However, a lot of cellular functions and responses that are present in natural tissues are lost in two-dimensional cell cultures. In this context, nanoparticle accumulation data presented in literature are often not accurate enough to predict behavior of nanoparticles in vivo. Cellular spheroids show a higher degree of morphological and functional similarity to the tissues. MethodsAccumulation and distribution of carboxylated CdSe/ZnS quantum dots (QDs), chosen as model nanoparticles, was investigated in cellular spheroids composed of different phenotype mammalian cells. The findings were compared with the results obtained in in vivo experiments with human tumor xenografts in immunodeficient mice. The diffusive transport model was used for theoretical nanoparticles distribution estimation. ResultsQDs were accumulated only in cells, which were localized in the periphery of cellular spheroids. CdSe/ZnS QDs were shown to be stable and inert; they did not have any side-effects for cellular spheroids formation. Penetration of QDs in both cellular spheroids and in vivo tumor model was limited. The mathematical model confirmed the experimental results: nanoparticles penetrated only 25μm into cellular spheroids after 24h of incubation. ConclusionsPenetration of negatively charged nanoparticles is limited not only in tumor tissue, but also in cellular spheroids. General SignificanceThe results presented in this paper show the superior applicability of cellular spheroids to cell monolayers in the studies of the antitumor effect and penetration of nanomedicines.

Constitutive IDO1 Expression in Human Tumors Is Driven by Cyclooxygenase-2 and Mediates Intrinsic Immune Resistance.

Tumors use various mechanisms to avoid immune destruction. Cyclooxygenase-2 (COX-2) expression may be a driver of immune suppression in melanoma, but the mechanisms involved remain elusive. Here, we show that COX-2 expression drives constitutive expression of indoleamine 2,3-dioxygenase 1 (IDO1) in human tumor cells. IDO1 is an immunosuppressive enzyme that degrades tryptophan. In a series of seven human tumor lines, constitutive IDO1 expression depends on COX-2 and prostaglandin E2 (PGE2), which, upon autocrine signaling through the EP receptor, activates IDO1 via the PKC and PI3K pathways. COX-2 expression itself depends on the MAPK pathway, which therefore indirectly controls IDO1 expression. Most of these tumors carry PI3K or MAPK oncogenic mutations, which may favor constitutive IDO1 expression. Celecoxib treatment promoted immune rejection of IDO1-expressing human tumor xenografts in immunodeficient mice reconstituted with human allogeneic lymphocytes. This effect was associated with a reduced expression of IDO1 in those ovarian SKOV3 tumors and an increased infiltration of CD3+ and CD8+ cells. Our results highlight the role of COX-2 in constitutive IDO1 expression by human tumors and substantiate the use of COX-2 inhibitors to improve the efficacy of cancer immunotherapy, by reducing constitutive IDO1 expression, which contributes to the lack of T-cell infiltration in "cold" tumors, which fail to respond to immunotherapy. Cancer Immunol Res; 5(8); 695-709. ©2017 AACR.

Efficacy of Anti-mesothelin Immunotoxin RG7787 plus Nab-Paclitaxel against Mesothelioma Patient-Derived Xenografts and Mesothelin as a Biomarker of Tumor Response.

Purpose: The purpose of this study was to evaluate the antitumor efficacy of the reduced immunogenicity anti-mesothelin immunotoxin RG7787 plus nab-paclitaxel against primary mesothelioma cell lines and tumor xenografts and the utility of mesothelin as a biomarker of tumor response.Experimental Design: Early-passage human malignant mesothelioma cell lines NCI-Meso16, NCI-Meso19, NCI-Meso21, and NCI-Meso29 were evaluated for sensitivity to RG7787 or nab-paclitaxel alone or in combination. In addition, the antitumor activity of RG7787 plus nab-paclitaxel was evaluated using NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumor xenografts in immunodeficient mice. Serum mesothelin was measured at different time points to determine whether its levels correlated with tumor response.Results: All four primary mesothelioma cell lines highly expressed mesothelin with 41 × 103 to 346 × 103 mesothelin sites per cell and were sensitive to RG7787, with IC50 ranging from 0.3 to 10 ng/mL. Except for NCI-Meso19, these cells were also sensitive to nab-paclitaxel, with IC50 of 10 to 25 ng/mL. In vitro, RG7787 plus nab-paclitaxel led to decreased cell viability compared with either agent alone. In NCI-Meso16 tumor xenografts, treatment with RG7787 plus nab-paclitaxel led to sustained complete tumor regressions. Similar antitumor efficacy was observed against NCI-Meso21 and NCI-Meso29 tumor xenografts. In all three tumor xenograft models, changes in human serum mesothelin correlated with response to therapy and were undetectable in mice with complete tumor regression with RG7787 and nab-paclitaxel.Conclusions: RG7787 plus nab-paclitaxel is very active against primary human mesothelioma cells in vitro as well as in vivo, with serum mesothelin levels correlating with tumor response. These results indicate that this combination could be useful for treating patients with mesothelioma. Clin Cancer Res; 23(6); 1564-74. ©2016 AACR.

Age and cellular context influence rectal prolapse formation in mice with caecal wall colorectal cancer xenografts.

In patients with rectal prolapse is the prevalence of colorectal cancer increased, suggesting that a colorectal tumor may induce rectal prolapse. Establishment of tumor xenografts in immunodeficient mice after orthotopic inoculations of human colorectal cancer cells into the caecal wall is a widely used approach for the study of human colorectal cancer progression and preclinical evaluation of therapeutics. Remarkably, 70% of young mice carrying a COLO320DM caecal tumor showed symptoms of intussusception of the large bowel associated with intestinal lumen obstruction and rectal prolapse. The quantity of the COLO320DM bioluminescent signal of the first three weeks post-inoculation predicts prolapse in young mice. Rectal prolapse was not observed in adult mice carrying a COLO320DM caecal tumor or young mice carrying a HT29 caecal tumor. In contrast to HT29 tumors, which showed local invasion and metastasis, COLO320DM tumors demonstrated a non-invasive tumor with pushing borders without presence of metastasis. In conclusion, rectal prolapse can be linked to a non-invasive, space-occupying COLO320DM tumor in the gastrointestinal tract of young immunodeficient mice. These data reveal a model that can clarify the association of patients showing rectal prolapse with colorectal cancer.

Abstract 4875: Developing tumor-localized, combination immunotherapies

Abstract Building on the recent clinical successes of checkpoint inhibitor antibodies, the field of cancer immunotherapy is now focussing on combination treatment regimens to further improve efficacy benefits to patients. However, combining such systemically dosed agents is associated with a number of challenges including enhanced side effect profiles and high costs. One strategy being explored to overcome such issues is to dose the therapeutics directly into the tumor rather than systemically but many tumors will not be accessible for this type of treatment. We have developed a broadly applicable vector platform system, based on the potent chimeric oncolytic group B adenovirus enadenotucirev (EnAd), for directing the efficient local production of a combination of immunotherapeutic agents selectively within the tumor. The versatility and fidelity of the platform has been exemplified by encoding up to three separate biomolecules in the same virus, including antibodies, cytokines, chemokines and tumor-associated antigens, without altering other virus properties. A systemic clinical dosing regimen has been established for EnAd, with data directly demonstrating selective virus delivery to and protein production from colorectal and other tumor types. The advantage of this approach is that immunotherapeutics encoded in the virus can be produced locally, both in tumors that are not directly injectable and in metastases, while minimising systemic off-target effects. A candidate virus NG-345 has been designed to produce a combination of three secreted immunomodulatory agents (human IFNá, MIP1á and Flt3L) aimed at enhancing the recruitment and activation of immune cells into tumor cell nests. We have shown that NG-345 retains the full oncolytic properties (potency and selectivity) of the parental EnAd virus, with infected human tumor cells producing high levels of all three cytokine/chemokines in the culture supernatants. Functional activity of individual encoded agents has also been demonstrated using relevant cell-based assays. EnAd is highly human-tumor selective and does not replicate, produce infectious progeny or express endogenously regulated transgenes in non-human cells. In vivo evaluation of immuno-modulatory activities of armed viruses is therefore challenging and requires the application of multiple approaches that can collectively provide informative data. In particular, studies are focusing on using surrogate candidate viruses expressing murine gene homologs in human tumor xenografts in immunodeficient mice with or without a reconstituted immune system. Citation Format: Brian R. Champion, Nalini Rasiah, Sam Illingworth, Matthieu Besneux, Rochelle Lear, Darren Plumb, Prithvi Kodialbail, Alice C.N. Brown. Developing tumor-localized, combination immunotherapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4875.

Abstract 630: Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice

Abstract Human tumor xenografts in immunodeficient mice have led to valuable insights into the biology of human cancer. The corresponding limitations and pitfalls of xenograft models have been described extensively. To tackle some limitations, we supplemented the murine tumor microenvironment (TME) with human stromal fibroblasts to mirror tumor-stroma cross-talk in vivo. Non-small cell lung cancer (NSCLC) cell lines Calu-1 and H1437 were co-injected with different types of fibroblasts into NOG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice in 3 independent experiments (n = 4-6/group; 106 in total). Human Dermal Fibroblasts (HDFs), lung cancer associated fibroblasts (CAFs), corresponding normal fibroblasts (NFs) and NSCLC cells were implanted into the lungs of 4-6 week old recipient mice. The influence of extracellular matrix components was evaluated by using either a matrigel/collagen mixture or alginate capsules. Tumor load was determined via overall survival (OS) and flow cytometry (FC). When mice had to be sacrificed, organs were collected and analyzed by FC and patho-histological examination. In a subsequent experiment, mice were sacrificed and tumors analyzed at defined time points and similar analyses performed. OS and metastatic pattern were similar in both cell lines when injected in mono-culture. OS of Calu-1 injected mice was not influenced when cells were co-injected with HDF but prolonged when co-injected with CAFs or NFs. In H1437 bearing mice, OS was significantly reduced when cells were co-injected with HDF or NF (Log-rank [Mantel-cox] test; p< 0.002 & 0.005), whereas CAFs had no influence on OS. Co-injection of HDF and Calu-1 enhanced the number of tumor cells in bronchoalveolar lavage fluid, whereas CAFs had the opposite effect (1way ANOVA for all following statistics, p< 0.0001). Consistently, the number of circulating Calu-1 cells was highest in co-culture with HDF and significantly lower in mice bearing Calu-1 and CAF or NF (p<0.0021). Co-injection of fibroblasts enhanced the H1437 tumor load in all compartments. Of note, the effect was not statistically significant. The use of alginate capsules was favorable compared to matrigel/collagen, improving fibroblast engraftment in all investigated organs. Furthermore, alginate capsules enhanced the number of circulating H1437 cells when co-injected with HDF as compared to matrigel/collagen (p<0.0001). Our results demonstrate the major impact of fibroblasts on tumor cell behavior in a preclinical setting. With the successful co-cultivation of human fibroblast and NSCLC cells in vivo, it will be possible to study tumor-stroma interactions in a clinically relevant mouse model. Once validated by a compound screening approach, this model will help to reduce drug failure rates and contribute to a more efficient development of urgently needed novel anti-cancer treatments. Citation Format: Eva Oswald, Vitor E. Santo, Albin Rudisch, Catarina Brito, Wolfgang Sommergruber, Helmut Dolznig, Julia B. Schüler. Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 630.