Trupti Vardam

Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells

Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers—CD34, neuropilin 1, and human kinase insert domain-containing receptor—and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach.

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Significance Inhibition of the VEGF/VEGF receptor (VEGFR) pathway has failed to increase overall survival in phase III trials in patients with glioblastoma (GBM). Previously we identified the angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway as a potential driver of resistance to VEGF inhibition in GBM. Here we show that dual inhibition of VEGFRs and Ang-2 inhibits tumor growth and prolongs vessel normalization compared with VEGFR inhibition alone, resulting in improved survival in murine GBM models. Furthermore, by blocking macrophage recruitment, we demonstrate that macrophages contribute to the beneficial effects of dual therapy. Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti–Ang-2–neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti–colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

Preclinical Efficacy of Ado-trastuzumab Emtansine in the Brain Microenvironment

BACKGROUND Central nervous system (CNS) metastases represent a major problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer because of the disappointing efficacy of HER2-targeted therapies against brain lesions. The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. METHODS We treated female nude mice bearing BT474 or MDA-MB-361 brain metastases (n = 9-11 per group) or cancer cells grown in organotypic brain slice cultures with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Data were analyzed with one-way analysis of variance (ANOVA), Kaplan-Meier analysis, and Coefficient of Determination. All statistical tests were two-sided. RESULTS T-DM1 delayed the growth of HER2-positive breast cancer brain metastases compared with trastuzumab. These findings were consistent between HER2-driven and PI3K-driven tumors. The activity of T-DM1 resulted in a survival benefit (median survival for BT474 tumors: 28 days for trastuzumab vs 112 days for T-DM1, hazard ratio = 6.2, 95% confidence interval = 6.1 to 85.84, P < .001). No difference in drug distribution or HER2-signaling was revealed between the two groups. However, T-DM1 led to a statistically significant increase in tumor cell apoptosis (one-way ANOVA for ApopTag, P < .001), which was associated with mitotic catastrophe. CONCLUSIONS T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. Clinical investigation of T-DM1 for patients with CNS metastases from HER2-positive breast cancer is warranted.

Obesity promotes resistance to anti-VEGF therapy in breast cancer by up-regulating IL-6 and potentially FGF-2

Targeting IL-6 and potentially FGF-2 overcomes resistance to anti-VEGF therapy in breast cancer. Tailored treatment for cancer in obesity Antiangiogenic therapy with inhibitors of the vascular endothelial growth factor (VEGF) has not proven effective in patients with many tumor types, including breast cancer. Meanwhile, obesity is a well-known risk factor in many cancer types, and once again, this includes breast cancer. Now, Incio et al. link these two observations, having discovered a mechanism by which obesity promotes resistance to VEGF inhibitor therapy through increased interleukin-6 and possibly also fibroblast growth factor 2 in the tumor microenvironment. The authors target these pathways in mouse models of cancer with and without obesity and demonstrate that the deleterious effects of obesity on VEGF inhibition can be overcome through the appropriate combination therapy. Anti–vascular endothelial growth factor (VEGF) therapy has failed to improve survival in patients with breast cancer (BC). Potential mechanisms of resistance to anti-VEGF therapy include the up-regulation of alternative angiogenic and proinflammatory factors. Obesity is associated with hypoxic adipose tissues, including those in the breast, resulting in increased production of some of the aforementioned factors. Hence, we hypothesized that obesity could contribute to anti-VEGF therapy’s lack of efficacy. We found that BC patients with obesity harbored increased systemic concentrations of interleukin-6 (IL-6) and/or fibroblast growth factor 2 (FGF-2), and their tumor vasculature was less sensitive to anti-VEGF treatment. Mouse models revealed that obesity impairs the effects of anti-VEGF on angiogenesis, tumor growth, and metastasis. In one murine BC model, obesity was associated with increased IL-6 production from adipocytes and myeloid cells within tumors. IL-6 blockade abrogated the obesity-induced resistance to anti-VEGF therapy in primary and metastatic sites by directly affecting tumor cell proliferation, normalizing tumor vasculature, alleviating hypoxia, and reducing immunosuppression. Similarly, in a second mouse model, where obesity was associated with increased FGF-2, normalization of FGF-2 expression by metformin or specific FGF receptor inhibition decreased vessel density and restored tumor sensitivity to anti-VEGF therapy in obese mice. Collectively, our data indicate that obesity fuels BC resistance to anti-VEGF therapy via the production of inflammatory and angiogenic factors.

Targeting the Tumor Microenvironment to Enhance Pediatric Brain Cancer Treatment.

Strategies targeting the microenvironment of pediatric brain cancers have the potential to improve the efficacy of standard and genome-based molecular therapeutics. These strategies also have the potential of helping resolve many of the challenges associated with developing new drugs and running clinical trials for relatively small pediatric brain tumor population. Disrupting vital paracrine and physical interactions between cancer cells and surrounding stroma, targeting and normalizing the abnormal tumor vasculature, and/or inducing antitumor immunity represent some of the most promising approaches. A comprehensive characterization of the pediatric brain tumor microenvironments composition and function and its modulation by chemoradiation and molecularly targeted therapies is warranted to develop and effectively implement these approaches.

Phase I and biomarker study of plerixafor and bevacizumab in recurrent high-grade glioma

Purpose: Although antiangiogenic therapy for high-grade glioma (HGG) is promising, responses are not durable. Correlative clinical studies suggest that the SDF-1α/CXCR4 axis may mediate resistance to VEGFR inhibition. Preclinical data have demonstrated that plerixafor (a reversible CXCR4 inhibitor) could inhibit glioma progression after anti-VEGF pathway inhibition. We conducted a phase I study to determine the safety of plerixafor and bevacizumab in recurrent HGG. Patients and Methods: Part 1 enrolled 23 patients with a 3 × 3 dose escalation design to a maximum planned dose of plerixafor 320 μg/kg subcutaneously on days 1 to 21 and bevacizumab 10 mg/kg intravenously on days 1 and 15 of each 28-day cycle. Cerebrospinal fluid (CSF) and plasma samples were obtained for pharmacokinetic analyses. Plasma and cellular biomarkers were evaluated before and after treatment. Part 2 enrolled 3 patients and was a surgical study to determine plerixafors penetration in tumor tissue. Results: In Part 1, no dose-limiting toxicities were seen at the maximum planned dose of plerixafor + bevacizumab. Treatment was well tolerated. After plerixafor 320 μg/kg treatment, the average CSF drug concentration was 26.8 ± 19.6 ng/mL. Plerixafor concentration in resected tumor tissue from patients pretreated with plerixafor was 10 to 12 μg/g. Circulating biomarker data indicated that plerixafor + bevacizumab induces rapid and persistent increases in plasma SDF-1α and placental growth factor. Progression-free survival correlated with pretreatment plasma soluble mesenchymal–epithelial transition receptor and sVEGFR1, and overall survival with the change during treatment in CD34+ progenitor/stem cells and CD8 T cells. Conclusions: Plerixafor + bevacizumab was well tolerated in HGG patients. Plerixafor distributed to both the CSF and brain tumor tissue, and treatment was associated with biomarker changes consistent with VEGF and CXCR4 inhibition. Clin Cancer Res; 24(19); 4643–9. ©2018 AACR.

Abstract LB-346: Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

OBJECTIVE: Here we aimed to overcome resistance to anti-VEGF therapy in glioblastoma (GBM), the most common and aggressive adult primary brain tumor. INTRODUCTION: Current standard of care, including chemo-radiation, confers modest overall survival benefits of less than 1.5 years in patients. GBMs are highly dependent on angiogenesis, and anti-VEGF therapy is a promising approach to prolong survival. While anti-VEGF therapy prolongs progression-free survival in GBM, patients rapidly become refractory and overall survival is not increased. We previously demonstrated that angiopoietin-2 (Ang-2) may confer resistance to anti-VEGF receptor (VEGFR) treatment, as ectopic overexpression of Ang-2 compromises the benefits of VEGFR inhibition in murine GBM. Additionally, circulating Ang-2 levels in GBM patients rebound after an initial decrease following administration of cediranib, a pan-VEGFR tyrosine kinase inhibitor, suggesting Ang-2 may mediate resistance to anti-VEGF pathway inhibition. EXPERIMENTAL DESIGN: In two orthotopic models of GBM, Gl261 (murine) and U87 (human), we used MRI and Optical Frequency Domain Imaging (OFDI) to measure whether dual inhibition of Ang-2 and VEGFR reduces tumor burden and improves survival. We examined morphological changes in the tumor vasculature by immunohistochemistry, and phenotypical changes in tumor associated macrophages (TAMs) by flow cytometry. RESULTS: Dual therapy with cediranib (a pan-VEGFRs inhibitor) and MEDI3617 (an Ang-2 neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth. In U87 tumors, combined treatment increased tumor necrosis. Dual therapy enhanced morphological normalization of vessels and led to changes along the M1/M2 spectrum of CD45 + CD11b + F4/80 + TAMs. We analyzed the functional role of TAMs by inhibiting TAM recruitment with an anti-colony stimulating factor-1 antibody (anti-CSF-1) in combination with dual therapy. Depletion of TAMs compromised the survival benefit of anti-Ang-2/VEGFR therapy, suggesting that TAMs mediate the survival benefits of dual therapy. CONCLUSION: Here we successfully demonstrated that dual inhibition of Ang-2 and VEGFRs prolongs survival in preclinical GBM models by reducing the viable tumor burden, improving vascular normalization, and reshaping the tumor immune-microenvironment mediated by TAMs. This combination may represent a potential therapeutic strategy to overcome the limitations of anti-VEGF monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells. Citation Format: Zohreh Amoozgar, Teresa E. Peterson, Nathaniel D. Kirkpatrick, Yuhui Huang, Christian T. Farrar, Koen A. Marijt, Jonas Kloepper, Meenal Datta, Giorgio Seano, Keehoon Jung, Walid S. Kamoun, Trupti Vardam, Matija Snuderl, Jermaine Goveia, Sampurna Chatterjee, Ana Batista, Alona Muzikansky, Ching Ching Leow, Lei Xu, Tracy T. Batchelor, Dan G. Duda, Dai Fukumura, Jain K. Jain. Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages. [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 LB-346.

Abstract A12: Microenvironmental Interleukin-6 induced activation of STAT3 signaling in medulloblastoma: Implications for molecular pathology and therapy

Aberrant activation of Janus kinase (JAK) / signal transducer and activator of transcription (STAT) 3 pathway has been implicated in tumor initiation, progression and metastasis which make it a potential therapeutic target in human malignancies. In vitro STAT3 inhibition exhibited anti-tumor effect in medulloblastoma (MB) cell lines; however, little has been reported about the mechanisms of STAT3 activation in medulloblastoma (MB). We found that STAT3 was constitutively activated in both medulloblastoma patient samples and 3 mice models that were clinically classified as SHH subgroup, Group 3 and Group 4 in pediatric MB patients. By using PCR arrays we investigated the early expression profile of local cytokines in host cerebellum during MB pathogenesis and found increased IL17B, IL6 and TNFα in the microenvironment of MB mice models. In vitro cytokine stimulation assay indicated that IL6 was the major activator of STAT3 in D283 MB cells. Confocal immunofluorescence staining of different cell components in the cerebellum in D283 xenografts showed that the endothelial cells and Purkinje cells were the two major sources of IL6 secretion. Our data provides molecular and cellular clue for the activation of STAT3 signaling during MB tumorigenesis and might help to develop more effective therapeutic targets for STAT3 blockade in MB. Citation Format: Shuang Yan, Sampurna Chatterjee, Trupti Vardam, Shuji Kitahara, Tai Hato, Sylvie Roberge, Vasileios Askoxylakis, Mark Duquette, Dai Fukumura, Lei Xu, Rakesh Jain. Microenvironmental Interleukin-6 induced activation of STAT3 signaling in medulloblastoma: Implications for molecular pathology and therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A12.

Abstract B70: Combination of AT1R blockade with CD40 activation provides enhanced therapeutic efficacy for mouse pancreatic adenocarcinoma

Pancreatic ductal adenocarcinomas (PDAC) have a dense stroma -- rich in type I collagen and hyaluronan -- which compresses blood vessels thus reducing vascular perfusion, and the delivery and efficacy of drugs. In parallel, the PDAC microenvironment is immunosuppressive. Therefore, one major goal of our current studies is to determine how the targeting of tumor stroma and immune modulation affects the efficacy of pancreatic cancer therapy. CD40 activation by agonist antibodies can increase the infiltration of tumoricidal macrophages in PDAC models and patients. PDAC desmoplasia is driven by tumor-associated fibroblasts, which are activated by downstream effectors of angiotensin II. Our group has previously shown that the angiotensin II type I receptor (AT1R) blocker losartan reduces desmoplasia and improves the delivery and effectiveness of cytotoxic agents in PDAC models. Moreover, the AT1R blocker telmisartan has potent anti-fibrotic effects at doses that do not affect the mean arterial blood pressure in mice. Also compared to losartan, telmisartan has a greater volume of distribution in tissue. In the present study we determined in a murine model of PDAC how the CD40 agonist antibody FGK45 and telmisartan affect the tumor stroma, the infiltration of immune cells and tumor growth. We first analyzed the effects of CD40-activation by the agonist antibody FGK45 (100 µg / mouse, iv every other day for 8 days) and telmisartan (1 or 5 mg / kg / day) on the tumor stroma in the orthotopic PDAC model AK4.4. FGK45 did not modify the tumor area occupied by collagen, but significantly reduced the tumor area occupied by hyaluronan. To assess vessel perfusion, we injected fluorescent-lectin intravenously then sacrificed mice 5 min later. In comparison to vehicle-treated tumors there was a trend for a lower density of CD31-positive vessels in telmisartan-treated tumors, however the difference was not significant. Telmisartan did not affect the fraction of lectin-perfused vessels. FGK45 did not change the density of CD31-positive vessels or the number of lectin-perfused vessels. We also determined the effects of a higher dose of telmisartan (10 mg / kg /day) and telmisartan combined with FGK45 on tumor perfusion and hypoxia. Pimonidazole and biotin-lectin were administered respectively 1 hour and 5 min before tumor resection. The higher dose of telmisartan alone or combined with FGK45 significantly increased vascular perfusion. In tumors treated with telmisartan alone or combined with FGK45 there was also a trend for a decrease in hypoxia. We analyzed the composition of the tumor immune infiltrate by flow cytometry. Both FGK45 and FGK45 combined with telmisartan significantly increased the infiltration of macrophages in tumors and reduced the recruitment of monocytic myeloid derived suppressor cells. Interestingly, FGK45 induced a significant increase in B cell infiltration in AK4.4 tumors. We then determined the effects of the FGK45 antibody and telmisartan on tumor growth. We treated mice with AK4.4 tumors with four doses (100 µg / mouse, iv every other day) of FGK45. In mice treated with FGK45 the AK4.4 tumors were significantly smaller than in the control group. To determine the effects of telmisartan on tumor growth, mice with AK4.4 tumors were surgically implanted with ALZET osmotic pumps and continuously dosed with telmisartan (1 or 5 mg / kg / day) for 12 days. Interestingly, both the dose of 1 and 5 mg / kg / day significantly reduced the growth of AK4.4 tumors. In contrast, in our previous studies with the same tumor model higher doses of losartan (20 or 40 mg / kg / day) did not affect tumor growth. We also evaluated the effect of FGK45 combined with telmisartan. Mice were continuously dosed with telmisartan (10 mg / kg / day) or the DMSO vehicle by osmotic pumps. Two days after the start of telmisartan treatment, 3 doses of FGK45 or a control IgG2a antibody were administered (100 µg / mouse, IV, every other day). We observed tumor regression after FGK45 treatment in 1 of 8 mice and after FGK45 combined with telmisartan treatment in 2 of 7 mice. We also found that both FGK45 alone or combined with telmisartan significantly reduced the bloody ascites, a hallmark of metastasis. In conclusion, our findings show that CD40 activation improves the infiltration of macrophages and B cells in PDAC lesions. While CD40 activation significantly reduced the accumulation of hyaluronan, it did not affect vascular perfusion or hypoxia. In contrast, telmisartan alone or combined with FGK45 increased vascular perfusion and reduced hypoxia, and did not affect the immune infiltration caused by CD40 activation. FGK45-activation of CD40 alone or combined with telmisartan induced tumor regressions. Currently we are evaluating the effects of FGK45 combined with telmisartan on mice survival. Citation Format: Shiwei Han, Jelena Grahovac, Trupti Vardam, Yves Boucher. Combination of AT1R blockade with CD40 activation provides enhanced therapeutic efficacy for mouse pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B70.