Changes In Tumor Cell Proliferation Research Articles

CCR7/DUSP1 signaling Axis mediates iCAF to regulates head and neck squamous cell carcinoma growth

ObjectiveC-C motif chemokine receptor 7 (CCR7) significantly influences tumors onset and progression, yet its impact on the tumor microenvironment (TME) and specific mechanisms remain elusive. Inflammatory Cancer-Associated Fibroblasts (iCAF), a vital subtype of Cancer-Associated Fibroblasts (CAF), play a critical role in regulating the TME and tumor growth, though the underlying molecular mechanisms are not fully understood. This study aims to determine whether CCR7 participates in tumor regulation by iCAF and to elucidate the specific mechanisms involved. MethodsDifferential gene analysis of CAF subtypes in CCR7 knockout and wild-type groups was conducted using single-cell data. Animal models facilitated the extraction of primary iCAF cells via flow cytometry sorting. Changes in DUSP1 expression and the efficiency of lentivirus-mediated knockdown and overexpression were examined through qPCR and Western Blot. MOC1 and MOC2 cells were co-cultured with iCAF, with subsequent validation of changes in tumor cell proliferation, migration, and invasion using CCK8, EdU, and wound healing assays. ELISA was employed to detect changes in TGF-β1 concentration in the iCAF supernatant. ResultsCAF was categorized into three subtypes—myCAF, iCAF, and apCAF—based on single-cell data. Analysis revealed a significant increase in DUSP1 expression in iCAF from the CCR7 knockout group, confirmed by in vitro experiments. Co-culturing MOC1 and MOC2 cells with iCAF exhibiting lentivirus-mediated DUSP1 knockdown resulted in inhibited tumor cell proliferation, invasion, and migration. In contrast, co-culture with iCAF overexpressing DUSP1 enhanced these capabilities. Additionally, the TGF-β1 concentration in the supernatant increased in the DUSP1 knockdown iCAF group, whereas it decreased in the DUSP1 overexpression group. ConclusionThe CCR7/DUSP1 signaling axis regulates tumor growth by modulating TGF-β1 secretion in iCAF.

Neoadjuvant Exercise Therapy in Prostate Cancer

Observational data have shown that postdiagnosis exercise is associated with reduced risk of prostate cancer death. The feasibility and tumor biological activity of exercise therapy is not known. To identify recommended phase 2 dose of exercise therapy for patients with prostate cancer. This single-center, phase 1a dose-finding trial was conducted at a tertiary cancer center using a patientcentric, decentralized platform and included 53 inactive men with treatment-naive localized prostate cancer scheduled to undergo surgical resection between June 2019 and January 2023. Data were analyzed in June 2024. Six escalated exercise therapy dose levels ranging from 90 to 450 minutes per week of individualized, moderate-intensity treadmill walking, allocated using adaptive continual reassessment. All exercise therapy sessions were conducted remotely with real-time monitoring. Feasibility was evaluated by relative exercise dose intensity (REDI). A dose level was considered feasible if 70% or more of patients achieved an REDI of 75% or greater. Activity end points were changes in tumor cell proliferation (Ki67) and plasma prostate-specific antigen levels between pretreatment and postintervention. Safety and changes in patient physiology were also assessed. A total of 53 men were enrolled (median [IQR] age, 61 [56-66] years). All dose levels were feasible (≥75% REDI). The mean (95% CI) changes in Ki67 were 5.0% (-4.3% to 14.0%) for 90 minutes per week, 2.4% (-1.3% to 6.2%) for 150 minutes per week, -1.3% (-5.8% to 3.3%) for 225 minutes per week, -0.2% (-4.0% to 3.7%) for 300 minutes per week, -2.6% (-9.2% to 4.1%) for 375 minutes per week, and 2.2% (-0.8% to 5.1%) for 450 minutes per week. Changes in prostate-specific antigen levels were 1.0 ng/mL (-1.8 to 3.8) for 90 minutes per week, 0.2 ng/mL (-1.1 to 1.5) for 150 minutes per week, -0.5 ng/mL (-1.2 to 0.3) for 225 minutes per week, -0.2 (-1.7 to 1.3) for 300 minutes per week, -0.7 ng/mL (-1.7 to 0.4) for 375 minutes per week, and -0.9 ng/mL (-2.4 to 0.7) for 450 minutes per week. No serious adverse events were observed. Overall, 225 minutes per week (approximately 45 minutes per treatment at 5 times weekly) was selected as the recommended phase 2 dose. The results of this nonrandomized clinical trial suggest that neoadjuvant exercise therapy is feasible and safe with promising activity in localized prostate cancer. ClinicalTrials.gov Identifier: NCT03813615.

LncRNA HCG18 promotes prostate cancer progression by regulating the miR-512-3p/HK-2 axis

ObjectiveLong non-coding RNAs (lncRNAs) play an important role in tumor progression. Numerous studies show that lncRNAs are strongly associated with prostate cancer (PCa) progression. The aim of this study was to investigate the pathway through which lncRNA HCG18 regulates PCa progression by bioinformatics analysis and experiments. MethodsWe compared HCG18 expression in PCa versus normal tissue and cells by data and cell lines, followed by comparing the changes in tumor cell proliferation, migration, and invasive ability after knockdown of HCG18. Then we searched for its downstream pathway by database and validated the pathway in vivo and in vitro. ResultsHCG18 was highly expressed in PCa and has the ability to promote tumor proliferation, migration, and invasion; knockdown of HCG18 led to a decrease in the ability of cells to do so, which can be reversed by knockdown of miR-512-3p or overexpression of hexokinase 2. ConclusionOur in vivo and in vitro experiments suggest that HCG18 can play a role in promoting PCa progression by blocking the inhibition of hexokinase 2 by miR-512-3p via sponge adsorption.

Glucose-Thymidine Ratio as a Metabolism Index Using 18F-FDG and 18F-FLT PET Uptake as a Potential Imaging Biomarker for Evaluating Immune Checkpoint Inhibitor Therapy.

Immune checkpoint inhibitors (ICIs) are widely used in cancer immunotherapy, requiring effective methods for response monitoring. This study evaluated changes in 18F-2-fluoro-2-deoxy-D-glucose (FDG) and 18F-fluorothymidine (FLT) uptake by tumors following ICI treatment as potential imaging biomarkers in mice. Tumor uptakes of 18F-FDG and 18F-FLT were measured and compared between the ICI treatment and control groups. A combined imaging index of glucose–thymidine uptake ratio (GTR) was defined and compared between groups. In the ICI treatment group, tumor growth was effectively inhibited, and higher proportions of immune cells were observed. In the early phase, 18F-FDG uptake was higher in the treatment group, whereas 18F-FLT uptake was not different. There was no difference in 18F-FDG uptake between the two groups in the late phase. However, 18F-FLT uptake of the control group was markedly increased compared with the ICI treatment group. GTR was consistently higher in the ICI treatment group in the early and late phases. After ICI treatment, changes in tumor cell proliferation were observed with 18F-FLT, whereas 18F-FDG showed altered metabolism in both tumor and immune cells. A combination of 18F-FLT and 18F-FDG PET, such as GTR, is expected to serve as a potentially effective imaging biomarker for monitoring ICI treatment.

Abstract P2-08-10: First results of the randomized window of opportunity clinical trial D-Biomark: Immunomodulatory effect of denosumab in early breast cancer

Abstract Background: Most breast cancers (BC) exhibit low immune infiltration and are unresponsive to immunotherapy. Inhibitors of the Receptor Activator of NFkB (RANK) pathway, such as denosumab, used for the treatment of bone metastasis, have been shown to prevent BC, reducing tumor cell proliferation and survival. Our recent data supports that RANK pathway inhibition in BC cells enhances anti-tumor immune response. However, the population of BC patients who may benefit from denosumab remains to be identified. The aim of the study is to evaluate the antiproliferative, proapoptotic and/or immunomodulatory activity of denosumab in early BC and to identify biomarkers of response. Methods: Patients with early stage HER2-negative BC, candidates to tumor excision as first therapeutic approach were included. Patients were randomized 2: 1 to denosumab (two doses of 120mg/Kg subcutaneous denosumab, days 1 and 8 plus oral calcium 1000 mg daily for a month) and control arm (no treatment). Blood and tumor samples were collected at baseline and at surgery (2-4 weeks later). Putative changes in tumor cell proliferation by Ki67 immunohistochemistry (IHC), survival by Cleaved caspase-3 IHC and tumor infiltrating lymphocytes (TILs) quantified in H&E were evaluated between initial biopsy and surgery. RANK and RANKL expression will be analysed by IHC and the infiltrating immune populations will be characterized by specific antibodies (CD3, CD4, CD8, CD20, FoxP3, CD68, PDL1, PD1). We will analyse denosumab driven gene expression changes in tumor samples and tools such as CIBERSORT will be used to characterize the immune infiltrate. To assess the increase in TILs and the variation in Ki 67 and Cleaved caspase-3, the T-test for paired samples was used. A value of p less than 0.05 was defined as statistically significant. Result: We present results from the initial 45 patients enrolled out of 60, 31 cases in the experimental arm and 13 in the control arm. Mean age was 55,97 (range 37-88) years. 38 women with luminal breast cancer were analysed and 7 cases of triple negative BC (5 in experimental arm and 2 in control arm). Clinical and tumor characteristics were well balanced between both groups. No relevant toxicities were reported. There was no reduction of Ki67 in either of the two arms and no changes in Cleaved caspase-3 were observed. Interestingly, a statistically significant increase in TILs was observed in the denosumab treated group (p=0.0092, Paired t test) but not in the control group (p=0.68). 29.03% of patients treated with denosumab showed a ≥10% increase in TILs vs 7.14% in the control group (p=0.13). Denosumab was associated with an effective systemic inhibition of RANKL. No relationship was found between serum RANKL levels at baseline and response to denosumab. RNAseq analysis and immunophenotyping is still in progress. Conclusion: Short term neoadjuvant denosumab induces an immunomodulatory effect with an increase in stromal TILs in early BC. ClinicalTrials.gov Identifier: NCT03691311 Table 1.ResultsBiopsySurgeryKi 67 (mean frequency) Control24.5729.57Ki 67 (mean frequency) Experimental22.3927.90Cleaved caspase 3 (mean area) Control0.260.58Cleaved caspase 3 (mean area) Experimental0.350.42TILs (mean percentage) Control9.3410.29TILs (mean percentage) Experimental8.2913.54TILs incrementControl p=0.68 vs. Experimental p=0.009 Citation Format: Andrea Vethencourt, Eva M Trinidad, Anna Petit, María T Soler-Monsó, Clara Gómez Aleza, Ander Urruticochea, Amparo García-Tejedor, Anna Gumà Martinez, Veronica Obadia, Silvia Vazquez, Rafael Villanueva, Adela Fernánez, Monica Cejuela, Sabela Recalde Penabad, Agostina Stradella, Miguel Gil-Gil, Sonia Pernas, Eva Gonzalez-Suarez, Catalina Falo. First results of the randomized window of opportunity clinical trial D-Biomark: Immunomodulatory effect of denosumab in early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-10.

Role of Long Non-Coding RNA LINC00641 in Cancer.

Long non-coding RNAs (lncRNAs) are non-protein coding RNAs with more than 200 nucleic acids in length. When lncRNAs are located in the nucleus, they regulate chromosome structure, participate in chromatin remodeling, and act as transcription regulators. When lncRNAs are exported to the cytoplasm, they regulate mRNA stability, regulate translation, and interfere with post-translational modification. In recent years, more and more evidences have shown that lncRNA can regulate the biological processes of tumor proliferation, apoptosis, invasion and metastasis, and can participate in a variety of tumor signaling pathways. Long-gene non-protein coding RNA641 (LINC00641), located on human chromosome 14q11.2, is differentially expressed in a variety of tumors and is related to overall survival and prognosis, etc. Interfering the expression of LINC00641 can lead to changes in tumor cell proliferation, invasion, metastasis, apoptosis and other biological behaviors. Therefore, LINC00641 is a promising new biomarker and potential clinical therapeutic target. In this review, the biological functions, related mechanisms and clinical significance of LINC00641 in many human cancers are described in detail.

Inhibition of RANK signaling in breast cancer induces an anti-tumor immune response orchestrated by CD8+ T cells

Most breast cancers exhibit low immune infiltration and are unresponsive to immunotherapy. We hypothesized that inhibition of the receptor activator of nuclear factor-κB (RANK) signaling pathway may enhance immune activation. Here we report that loss of RANK signaling in mouse tumor cells increases leukocytes, lymphocytes, and CD8+ T cells, and reduces macrophage and neutrophil infiltration. CD8+ T cells mediate the attenuated tumor phenotype observed upon RANK loss, whereas neutrophils, supported by RANK-expressing tumor cells, induce immunosuppression. RANKL inhibition increases the anti-tumor effect of immunotherapies in breast cancer through a tumor cell mediated effect. Comparably, pre-operative single-agent denosumab in premenopausal early-stage breast cancer patients from the Phase-II D-BEYOND clinical trial (NCT01864798) is well tolerated, inhibits RANK pathway and increases tumor infiltrating lymphocytes and CD8+ T cells. Higher RANK signaling activation in tumors and serum RANKL levels at baseline predict these immune-modulatory effects. No changes in tumor cell proliferation (primary endpoint) or other secondary endpoints are observed. Overall, our preclinical and clinical findings reveal that tumor cells exploit RANK pathway as a mechanism to evade immune surveillance and support the use of RANK pathway inhibitors to prime luminal breast cancer for immunotherapy.

NUCLEAR MEDICINE TECHNIQUES FOR IN VIVO ANIMAL IMAGING

The object of the study was to analyze radionuclide detection techniques for in vivo animal imaging. Material and Methods. A total of 49 publications available from Scopus, Web of Science, Google Scholar eLIBRARY and Pubmed and published between 2013 and 2019 were reviewed. Results. The nuclear medicine techniques, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) are the most suitable imaging modalities for in vivo animal imaging. Besides traditional radiopharmaceuticals, such as [18F]-FDG and [99mTc]-MDP, the new radiolabeled tracers, such as [99mTc]-3PRGD2, [ 99mTc]-HisoDGR targeted to integrin, [18F]- tetrafluoroborate, labeled antibodies and others have been used for the noninvasive detection of tumors and for monitoring their response to treatment in mice and rats. 111In and 89Zr –labeled monoclonal antibodies are used to evaluate the expression level of many receptors such as EGFR, HER-2 and others in different tumors. PET imaging has demonstrated a good efficacy in tumor hypoxia imaging with [64Cu]-ATSM, [18F]-FMISO. PET and SPECT can also be used for early evaluation of anticancer therapy response. Nuclear imaging techniques may assist in the vivo assessment of DNA damage (doubleand single-strand brakes) as well as apoptosis intensity in tumor and normal tissues. [99mTc]- duramycin is the most commonly used tracer for imaging of apoptosis. Changes in tumor cell proliferation in response to anticancer therapy can be assessed by PET imaging with [18F]-FLT. Conclusion. Nuclear medicine offers a unique means to study cancer biology in vivo and to optimize cancer therapy.

Mesothelin Enhances Tumor Vascularity in Newly Forming Pancreatic Peritoneal Metastases.

Over 90% of pancreatic ductal adenocarcinomas (PDAC) express mesothelin (MSLN). Overexpression or knockdown of MSLN has been implicated in PDAC aggressiveness. This activity has been ascribed to MSLN-induced activation of MAPK or NF-κB signaling pathways and to interaction of MSLN with its only known binding partner, MUC16. Here, we used CRISPR/Cas9 gene editing to delete MSLN from PDAC, then restored expression of wild-type (WT) or Y318A mutant MSLN by viral transduction. We found that MSLN KO cells grew in culture and as subcutaneous tumors in mouse xenografts at the same rate as WT cells but formed intraperitoneal metastases poorly. Complementation with WT MSLN restored intraperitoneal growth, whereas complementation with Y318A mutant MSLN, which does not bind MUC16, was ineffective at enhancing growth in both MUC16(+) and MUC16(-) models. Restoration of WT MSLN did enhance growth but did not affect cell-to-cell binding, cell viability in suspension or signaling pathways previously identified as contributing to the protumorigenic effect of MSLN. RNA deep sequencing of tumor cells identified no changes in transcriptional profile that could explain the observed phenotype. Furthermore, no histologic changes in tumor cell proliferation or morphology were observed in mature tumors. Examination of nascent MSLN KO tumors revealed decreased microvascular density as intraperitoneal tumors were forming, followed by decreased proliferation, which resolved by 2 weeks postimplantation. These data support a model whereby MSLN expression by tumor cells contributes to metastatic colonization. IMPLICATIONS: MSLN confers a growth advantage to tumor cells during colonization of peritoneal metastasis. Therapeutic blockade of MSLN might limit peritoneal spread.

Dynamic 18F-FLT PET imaging of spatiotemporal changes in tumor cell proliferation and vasculature reveals the mechanistic actions of anti-angiogenic therapy

Anti-angiogenic therapies target tumor vasculature and tumor cells, thus a concurrent assessment of these targets would lead to a greater understanding of therapeutic resistance and facilitate development of improved therapeutic strategies. We utilize dynamic 3′-deoxy-3′-18F-fluorothymidine positron emission tomography (18F-FLT PET) scanning to concurrently assess changes in tumor cell proliferation and vasculature during anti-angiogenic therapy, providing insight into how these therapies may be used effectively with combination chemotherapy. Thirty-three patients with advanced solid malignancies underwent treatment with vascular endothelial growth factor receptor inhibitor (VEGFR-TKI) axitinib on an intermittent schedule (two-weeks-on/one-week-off). Patients had up to three dynamic 18F-FLT PET/CT scans: at baseline, after two weeks of continuous VEGFR-TKI treatment, and following a one week treatment break. 18F-FLT kinetics were analyzed using a two-tissue compartment kinetic model. Kinetic parameters Vb and K1 were extracted to quantify changes in tumor vasculature and the 18F-FLT flux constant Ki was calculated to quantify changes in tumor cell proliferation. Two weeks of continuous axitinib exposure led to decreases in Vb (median −21%, P = 0.07), K1 (median −39%, P < 0.01), and Ki (median −37%, P < 0.01), corresponding to diminished tumor vasculature and cell proliferation that may antagonize treatment with concurrent chemotherapy. Axitinib treatment breaks led to significant increases in Vb (median +42%, P < 0.01), K1 (median +46%, P < 0.01), and Ki (median +39%, P < 0.01) that is suggestive of an optimal time to schedule synergistic chemotherapy. Significant negative correlations (rho ⩽ −0.70, P < 0.01) were found between changes in tumor vasculature during axitinib exposure weeks and changes in tumor vasculature during treatment breaks. Imaging with dynamic 18F-FLT PET revealed new insights relating to the interplay of vascular and proliferative pharmacodynamics of axitinib therapy, facilitating a greater understanding of the mechanistic actions of VEGFR-TKIs. Increases in tumor vasculature and cell proliferation during VEGFR-TKI treatment breaks, suggests this period is an optimal time to schedule synergistic chemotherapy and warrants further investigation.

Pharmacodynamic study using FLT PET/CT in advanced solid malignancies treated with a sequential combination of X-82 and docetaxel.

A sequential approach, synchronizing cell-cycle specific chemotherapy during VEGFR-TKI treatment breaks, may improve the therapeutic index of this combination therapy. In this study we investigate the safety/tolerability and pharmacodynamic effects of docetaxel used in sequential combination with the novel VEGFR-TKI X-82. Patients with advanced solid malignancies underwent 21-day treatment cycles with X-82 administered daily on days 1-14, a treatment break on days 15-20, and docetaxel administered on day 21. Randomization was 1:1 to either a low-dose X-82 (200mg) or high-dose X-82 (400mg) arm. Patients were scheduled to undergo four 3'-deoxy-3'-18F-fluorothymidine (FLT) PET/CT scans to assess changes in tumor cell proliferation. PET standardized uptake values (SUV) were summarized for tumors and changes were assessed using mixed effects models. 14 patients were enrolled and treated with median 3.5 cycles (range 0-12). Three patients in the high-dose cohort (50%) and three patients in the low-dose cohort (38%) experienced at least one grade 3 adverse event during the study (infections, cytopenias, electrolyte abnormalities, and vascular complications). Four patients with 13 metastatic tumors underwent FLT PET/CT scanning. During the cycle 1 X-82 exposure period, tumor SUVmax decreased by - 11% (p = 0.04). After administration of docetaxel and the cycle 2 X-82 exposure period, tumor SUVmax decreased - 44% (p = 0.03). The sequential combination of X-82 and docetaxel was safe and led to diminished FLT uptake. Further, decrease in FLT uptake during cycle 2 (X-82 plus docetaxel) was greater than in cycle 1 (X-82 alone), suggesting sequential chemotherapy enhances the pharmacodynamic effect of therapy.

18F-FLT PET/CT imaging in patients with advanced solid malignancies treated with axitinib on an intermittent dosing regimen.

This study utilizes FLT PET/CT imaging to characterize changes in tumor cell proliferation and vasculature during intermittent treatment with VEGR-TKI axitinib. Patients with metastatic solid malignancies underwent 3-week treatment cycles with axitinib (7 and 5mg BID for safety and pharmacodynamic cohorts, respectively). Cycles consisted of 2weeks of treatment (dosing period) followed by a 1-week treatment break (washout period). Patients in the pharmacodynamic cohort had up to six FLT PET/CT scans (three scans in each cycle 1 and cycle 3) and had plasma VEGF concentrations measured at imaging timepoints. Changes in tumor SUVs and VEGF within and across drug cycles were investigated. Eight patients enrolled in the safety cohort where it was determined 7mg axitinib was not tolerable due to severe adverse events, including three patients who experienced significant hypertension and thrombovascular effects. Sixteen patients enrolled in the pharmacodynamic cohort demonstrated significant decreases in SUVs and increases in VEGF during dosing periods. This was followed by significant increases in SUVs and decreases in VEGF during drug washout periods. No significant differences in SUVs or VEGF were found when comparing cycle 1 with cycle 3. A mixed effects model demonstrated significant negative correlation between SUV and VEGF. Response to axitinib included diminished FLT uptake during dosing periods followed by increased FLT uptake during drug washout periods. These changes were not different when comparing treatment cycle 1 versus cycle 3, suggesting that the pharmacodynamic effect of intermittent axitinib is similar across multiple drug cycles.

Abstract 1161: COX-2 blockade improves efficacy of VEGF-targeting drugs

Abstract Efficacy of anti-angiogenic drugs is hampered by hypoxia-induced resistance. Because cyclooxygenase-2 (Cox-2) is upregulated in hypoxic conditions we analyzed mRNA expression levels of cyclooxygenase-1 (Cox-1) and Cox-2 in GFP+ FACS-sorted tumor cells from 4T1 tumors after treatment with anti-VEGFR2 antibodies (DC101) or with sunitinib. Cox-2 but not Cox-1 mRNA was upregulated by 2.3-fold upon anti-angiogenic treatment. In addition we found 5.2-fold increased prostaglandin E2 levels in 4T1 tumors after anti-angiogenic therapy. We hypothesized that concomitant blockade of Cox-2 could increase efficacy of anti-angiogenic agents. Therefore we treated 4T1 tumor-bearing mice with sunitinib or DC101 alone and in combination with acetylsalicyclic acid (ASA). We found that single treatment with ASA or angiogenesis inhibitors inhibited tumor growth and that combined inhibition of Cox-2 and VEGF(R) signaling exerted additive therapeutic efficacy (n=5; 1142±84 (ASS); 1148±78 (Sunitnib) vs. 63±5 mg (combination); p&amp;lt;0.0001). Similar effects were achieved upon combining the specific Cox2 inhibitor SC-236 with anti-angiogenic therapy (n=6; 985±147 (SC-326); 1123±61 (Sunitnib) vs. 680±76 mg (combination); p&amp;lt;0.05) and in the 6CCL4 orthotopic breast cancer model (data not shown). We carried out an extensive profiling of the tumor cells and their microenvironment upon concomitant blockade of Cox-2 and VEGF signaling in order to elucidate the underlying mechanism. We found no changes in tumor cell proliferation or upon combined Cox-2 inhibition and anti-angiogenic therapies. Also combined Cox-2 and VEGF-signaling inhibition did not change the quantitative composition of the inflammatory tumor infiltrate. However, upon analyzing polarization of FACS-sorted TAMs we found that mRNA of the M1 markers iNOS, MHCII, IL1β and TNFα were upregulated upon treatment with ASA alone and/or in combination with anti-angiogenic agents compared to controls or monotherapy with anti-angiogenic agents. In contrast, the M2 markers Arg1 and YM1 were downregulated upon treatment with Cox2-inhibitors and anti-angiogenic agents. Therefore, Cox-2 inhibitors skew TAMs towards an anti-tumoral M1 phenotype while the pro-angiogenic, tumor promoting M2-phenotype is suppressed. In addition, Cox-2 and PGE2 can promote tumor angiogenesis. This alternative pro-angiogenic pathway would be enhanced by increased Cox-2 expression and PGE2 levels and could contribute to resistance against anti-angiogenic treatments. In line with this hypothesis the MVD was decreased 4T1 tumors treated with combined Cox-2 and VEGF blockade compared to the respective monotherapy (n=7; 31±2.4 (Sunitinib); 28.58±0.83 (ASS) vs. 9.67±1.71 (combination); p&amp;lt;0.001). In conclusion concomitant Cox2 inhibition and anti-angiogenic therapies exert pronounced additive effects, which are at least partially due to increased M1 polarization and additive anti-angiogenic effects. Citation Format: Isabel Ben Batalla, Miguel Cubas-Cordova, Florian Udonta, Mark Wroblewski, Stefanie Sawall, Victoria Gensch, Klaus Pantel, Carsten Bokemeyer, Sonja Loges. COX-2 blockade improves efficacy of VEGF-targeting drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1161. doi:10.1158/1538-7445.AM2014-1161

Combined VEGF and CXCR4 antagonism targets the GBM stem cell population and synergistically improves survival in an intracranial mouse model of glioblastoma.

Glioblastoma recurrence involves the persistence of a subpopulation of cells with enhanced tumor-initiating capacity (TIC) that reside within the perivascular space, or niche (PVN). Anti-angiogenic therapies may prevent the formation of new PVN but have not prevented recurrence in clinical trials, suggesting they cannot abrogate TIC activity. We hypothesized that combining anti-angiogenic therapy with blockade of PVN function would have superior anti-tumor activity. We tested this hypothesis in an established intracranial xenograft model of GBM using a monoclonal antibody specific for murine and human VEGF (mcr84) and a Protein Epitope Mimetic (PEM) CXCR4 antagonist, POL5551. When doses of POL5551 were increased to overcome an mcr84-induced improvement in vascular barrier function, combinatorial therapy significantly inhibited intracranial tumor growth and improved survival. Anti-tumor activity was associated with significant changes in tumor cell proliferation and apoptosis, and a reduction in the numbers of perivascular cells expressing the TIC marker nestin. A direct effect on TICs was demonstrated for POL5551, but not mcr84, in three primary patient-derived GBM isolates. These findings indicate that targeting the structure and function of the PVN has superior anti-tumor effect and provide a strong rationale for clinical evaluation of POL5551 and Avastin in patients with GBM.

Response Prediction to Neoadjuvant Chemotherapy: Comparison between Pre-Therapeutic Gene Expression Profiles and In Vitro Chemosensitivity Assay

Although the use of (neo-)adjuvant chemotherapy in breast cancer patients has resulted in improved outcome, not all patients benefit equally. We have evaluated the utility of an in vitro chemosensitivity assay in predicting response to neoadjuvant chemotherapy. Pre-therapeutic biopsies were obtained from 30 breast cancer patients assigned to neoadjuvant epirubicin 75 mg/m2 and docetaxel 75 mg/m2 (Epi/Doc) in a prospectively randomized clinical trial. Biopsies were subjected to a standardized ATP-based Epi/Doc chemosensitivity assay, and to gene expression profiling. Patients then received 3 cycles of chemotherapy, and response was evaluated by changes in tumor diameter and Ki67 expression. The efficacy of Epi/Doc in vitro was correlated with differential changes in tumor cell proliferation in response to Epi/Doc in vivo (p = 0.0011; r = 0.73670, Spearmańs rho), but did not predict for changes in tumor size. While a pre-therapeutic gene expression signature identified tumors with a clinical response to Epi/Doc, no such signature could be found for tumors that responded to Epi/Doc in vitro, or tumors in which Epi/Doc exerted an antiproliferative effect in vivo. This is the first prospective clinical trial to demonstrate the utility of a standardized in vitro chemosensitivity assay in predicting the individual biological response to chemotherapy in breast cancer.

Abstract C59: Antibody targeting of CD24 efficiently retards growth of experimental carcinomas.

Abstract It has been shown that CD24 drives lung metastasis formation of bladder cancer and might be a good candidate for mAb-dependent therapy. We tested whether mAb SWA11 to CD24 would be effective in preventing tumor growth in xenotransplanted mice. In an A549 lung carcinoma model cells were implanted s.c. into SCID/beige mice followed by multiple injections of mAb over 37 days. Therapy with anti-CD24 mAb SWA11 lead to substantially reduced tumor load. The final tumor mass of treated animals was significantly smaller compared to isotype-control mAb treated animals. Similar observations were made in therapy models of CD24-positive BxPC3 pancreatic adenocarcioma cells in NOD/SCID mice and SKOV-3ip ovarian carcinoma cells in CD1 mice. The therapeutic effects were associated with changes in tumor cell proliferation, apoptosis and angiogenesis. Surprisingly, we found that SWA11 mAb treatment significantly altered cytokine tumor microenvironment. Also the mAb treatment lead to the increased infiltration of tumor tissue with immune cells suggesting involvement of ADCC. Addition of SWA11 mAb to the gemcitabine treatment strongly potentiated its anti-cancer efficacy in A549 lung cancer model. Our data demonstrate that targeting of CD24 could be beneficial for the anti-cancer treatment using standard chemotherapy regimes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C59.

Abstract P2-09-20: Response Prediction to Neoadjuvant Chemotherapy: Comparison between Pre-Therapeutic Gene Expression Profiles and In Vitro Chemosensitivity Assay

Abstract Purpose: Although the use of (neo-)adjuvant chemotherapy in breast cancer patients has resulted in improved disease-free and overall survival, not all patients benefit equally. Nevertheless, chemotherapy is still applied empirically, since clinical tests for response prediction are not routinely available. We have evaluated the utility of an in vitro chemosensitivity assay (ATP-TCA) in predicting response to neoadjuvant chemotherapy. Methods: Pre-therapeutic tumor core biopsies were obtained from 65 breast cancer patients and subjected to a standardized ATP-based Epi/Doc tumor chemosensitivity assay (ATP-TCA). Of these, 30 breast cancer patients were also assigned to three cycles of neoadjuvant epirubicin 75 mg/m2 and docetaxel 75 mg/m2 (Epi/Doc) in a prospectively randomized clinical trial. Tumor response was evaluated by pre-and post-interventional ultrasound measurements of tumor diameter, and by differential Ki67 expression. Results: 3 Cycles of neoadjuvant Epi/Doc resulted in a clinical response according to RECIST criteria in 11/23 evaluable patients (48%), and in a profound reduction in tumor proliferation in 10/23 patients (43%). Similarly, in vitro treatment of tumor cell suspensions with Epi/Doc performed in 65 pre-treatment core-biopsies yielded chemosensitivity in comparable fraction of patients (30/65, 46%). When the differentail changes in tumor cell proliferation in response to 3 cycles Epi/Doc in 23 patients were compared with ATP-TCA results obtained from the pre-interventional tumor core-biopsies of the same individual patients, we found that the efficacy of Epi/Doc in vitro was significantly correlated with differential changes in tumor cell proliferation in response to 3 cycles Epi/Doc in vivo (p= 0.0011; r=0.73670, Spearman's rho), but did not predict for changes in tumor size when measured by ultrasound. Conclusion: This is the first prospective clinical trial to demonstrate the utility of a standardized in vitro chemosensitivity assay in predicting the individual response to chemotherapy in breast cancer patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-20.

MO-E-BRB-05: A Computational Tumor Modeling Framework for the Optimization of Molecular Targeted Therapies

Purpose: Current treatment schedules of molecular targeted therapies are established through costly clinical trials. Although several dosing schemes are used clinically, optimal dosing regimens remain unknown. We developed a computational tumor modeling framework to compare dosing schedules based on simulated therapeutic response. Method and Materials: A pharmacokinetic/pharmacodynamic model was developed to simulate changes in tumorcell proliferation and vascular function. The model was applied to data from a clinical trial in which patients received sunitinib, a molecular targeted agent with anti‐angiogenic and anti‐proliferative effects, on a 4/2 (4 weeks on, 2 week break) or 2/1 schedule. Using [18F]FLT PET/CT imaging, levels of tumor proliferation and vascular function were assessed at baseline, peak drug exposure, and during treatment break. After testing the model on data from the 4/2 schedule, we compared simulated therapeutic responses for these dosing regimens: 4/2 cycle, two consecutive 2/1 cycles, and continuous dosing. Results: Trends in proliferative response were successfully simulated within one standard error of the population means. Two consecutive 2/1 cycles resulted in a 12% greater decrease in tumor proliferation as compared to one 4/2 cycle due to decreased drug washout during the off‐drug period. For iso‐response conditions, the dose for the 2/1 schedule could be reduced to 80% of the 4/2 schedule (from 50mg/kg/day to 40mg/kg/day). Continuous dosing using lower daily doses (32.5mg/kg/day) yielded the best growth inhibition after 6 weeks. Conclusion: The implemented model successfully reproduced trends in proliferative response observed in patients receiving sunitinib. Continuous dosing yielded the best growth inhibition, and outperformed two consecutive 2/1 cycles and the 4/2 regimen, indicating that this regimen might be favorable, especially for patients requiring lower daily doses to manage toxic side effects. Upon successful validation, the implemented model could serve as a cost‐effective tool to help identify improved drug regimens.

Response prediction to neoadjuvant chemotherapy: Comparison between pretherapeutic gene expression profiles and in vitro chemosensitivity assay.

10516 Background: Although the use of (neo-)adjuvant chemotherapy in breast cancer patients has resulted in improved disease-free and overall survival, not all patients benefit equally. Nevertheless, chemotherapy is still applied empirically, since clinical tests for response prediction are not routinely available. We have evaluated the utility of an in vitro chemosensitivity assay (ATP-TCA) and gene expression profiles in predicting response to neoadjuvant chemotherapy. Methods: Pre-therapeutic core biopsies were obtained from 30 breast cancer patients assigned to three cycles of neoadjuvant epirubicin 75 mg/m2 and docetaxel 75 mg/m2 (Epi/Doc) in a prospectively randomized clinical trial (ABCSG 14). Individual tumor samples were subjected to a standardized ATP-based Epi/Doc tumor chemosensitivity assay (ATP-TCA), and to Affymetrix U133 A+B gene expression arrays. Patients then received 3 cycles of chemotherapy, and tumor response was subsequently evaluated by pre-operative ultrasound and Ki67 expression. Results: The efficacy of Epi/Doc in vitro was significantly correlated with differential changes in tumor cell proliferation in response to 3 cycles Epi/Doc in vivo (p = 0.0011; r = 0.73670, Spearmanxs rho), but did not predict for changes in tumor size when measured by ultrasound. While a pre-therapeutic gene expression signature identified tumors with a clinical response to Epi/Doc, no such signature could be found for tumors that responded well to Epi/Doc in vitro, or tumors in which Epi/Doc exerted a strong antiproliferative effect in vivo. Conclusions: This is the first prospective clinical trial to demonstrate the utility of a standardized in vitro chemosensitivity assay in predicting the individual response to chemotherapy in breast cancer patients. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration DCS Diagnostica GlaxoSmithKline, Novartis, Roche

Edema Control by Cediranib, a Vascular Endothelial Growth Factor Receptor–Targeted Kinase Inhibitor, Prolongs Survival Despite Persistent Brain Tumor Growth in Mice

Recent clinical trials of antivascular endothelial growth factor (VEGF) agents for glioblastoma showed promising progression-free and overall survival rates. However, available clinical imaging does not separate antitumor effects from antipermeability effects of these agents. Thus although anti-VEGF agents may decrease tumor contrast-enhancement, vascularity, and edema, the mechanisms leading to improved survival in patients remain incompletely understood. Our goal was to determine whether alleviation of edema by anti-VEGF agents alone could increase survival in mice. We treated mice bearing three different orthotopic models of glioblastoma with a VEGF-targeted kinase inhibitor, cediranib. Using intravital microscopy, molecular techniques, and magnetic resonance imaging (MRI), we measured survival, tumor growth, edema, vascular morphology and function, cancer cell apoptosis and proliferation, and circulating angiogenic biomarkers. We show by intravital microscopy that cediranib significantly decreased tumor vessel permeability and diameter. Moreover, cediranib treatment induced normalization of perivascular cell coverage and thinning of the basement membrane, as mirrored by an increase in plasma collagen IV. These rapid changes in tumor vascular morphology and function led to edema alleviation -- as measured by MRI and by dry/wet weight measurement of water content -- but did not affect tumor growth. By immunohistochemistry, we found a transient decrease in macrophage infiltration and significant but minor changes in tumor cell proliferation and apoptosis. Systemically, cediranib increased plasma VEGF and placenta growth factor levels, and the number of circulating CXCR4(+)CD45(+) cells. However, by controlling edema, cediranib significantly increased survival of mice in the face of persistent tumor growth. Anti-VEGF agents may be able to improve survival of patients with glioblastoma, even without inhibiting tumor growth.