Non-tumor Bearing Animals Research Articles

Abstract 3206: Tumor-targeted nanoparticles for cancer therapy

Abstract Introduction: Untargeted cancer therapy is often toxic, limiting the maximum tolerated dose (MTD) and thus potential cure. Targeted therapy with antibody-drug conjugates (ADCs) has demonstrated greater efficacy, as with those directed at HER2 for triple negative breast cancer and other HER2 expressing tumors. However, ADC drug-antibody ratio (DAR) is limited, typically 3 to 1. In contrast, antibody-targeted nanoparticles deliver thousands of drug molecules per nanoparticle, with potentially greater anti-tumor effect. Experimental Procedures: Antibody targeted nanoparticles (TNP) were created that preferentially deliver cytotoxic molecules to tumor cells. TNPs were created with various antibodies and payloads and evaluated for their efficacy against 12 different adult and pediatric malignancies (see below), using tumor xenografts in NOG-SCID mice. Survival of control, free drug, and TNP treated animals was compared. MED was also determined. Non-tumor bearing animals treated at the same dose and schedule were evaluated for evidence of toxicity. PK/PD metrics were obtained and drug delivery to normal and tumor tissue was evaluated microscopically and by mass spectroscopy. Data: Plasma TNP drug levels persist for 48 hours, compared to free drug which is undetectable by 2 hours. Microscopically, nanoparticles accumulate in tumor tumor tissue but not normal tissue. Drug levels are typically 15 times higher in tumor tissue than normal tissue by quantitative mass spectroscopy. The minimum effective dose (MED) is reduced 25-fold for Ewing sarcoma, from 50 mg/kg to 2 mg/kg. TNPs also cross the blood-brain barrier (BBB), and were used to treat orthotopic GBM PDX xenografts, prolonging animal survival 3-fold over control or free drug treated animals. Ewing sarcoma, osteosarcoma, triple negative breast cancer, GBM, hepatoma, and adult ALL, bearing animals all showed complete tumor ablation and 100% survival when treated biweekly at 10mg/kg. Pancreatic, prostate, and ovarian Ca showed 100% survival in TNP treated mice after all control animals had died but remained tumor bearing with slow but progressive tumor growth when treated identically. Lung and colorectal Ca and melanoma bearing animals treated similarly with TNPs showed 50-80% survival at the time of control animal death. No animal in any study showed evidence of off-target toxicity. Conclusions: Tumor antigen targeted, cytotoxic molecule containing nanoparticles have been created that show broad utility in the treatment of at least a dozen different adult and pediatric cancers. Six of 12 types showed complete tumor ablation when treated at 10mg/kg biweekly injections, and the remainder showed enhanced anti-tumor effect and survival compared to free drug treatment. We conclude that use of tumor antigen targeted, small molecule cytotoxic containing nanoparticles results in dramatically enhanced tumor response and in many case, complete tumor ablation where the same free drug fails to do so. Citation Format: HyungGyoo Kang, Bryon Upton, Jon Nagy, Timothy Triche. Tumor-targeted nanoparticles for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3206.

Host B cells escape CAR-T immunotherapy by reversible downregulation of CD19.

Anti-CD19-CAR-T cells are a successful clinical immunotherapy for B cell lymphomas, although some lymphomas can escape attack by downregulating surface CD19 levels. An undesirable consequence of this therapy is that it can also eliminate healthy B cells expressing CD19. Therefore, understanding the dynamics of CD19 expression in B cells under CAR-T cell immunotherapy can help mitigate both escape and adverse outcomes. Previous studies suggested that mechanisms responsible for the loss of CD19 expression in lymphomas usually involves genomic deletion or epigenetic modification which permanently removes CD19 as a therapeutic target in these cells. We examined if healthy B cells can use similar processes to lose CD19 expression and escape CAR-T attack. In the presence of CAR-T cells, untransformed B cells both when cultured in vitro or in vivo in non-tumor bearing animals downregulate expression of CD19. We then used adoptive transfer strategies to remove CD19-low B cells from αCD19-CAR-T pressure in vivo. Intriguingly, these B cells systematically recovered surface expression of CD19 comparable to wild-type levels. These data suggest that unlike many cases of lymphomas, healthy B cells downregulate CD19 in a reversible fashion. Taken together, these data suggest a dynamic regulatory process of CD19 surface expression on healthy B cells that could be exploited to modulate the expression of CD19 on cancer cells to improve immunotherapy or minimize the depletion of endogenous B cell compartment during treatment.

Abstract 2548: Preclinical development of EARLI-001, a genetic platform producing cancer-activated synthetic biomarkers for the early detection of malignancies

Abstract Early detection of cancer is a powerful driver of increased survival rate, quality of life and reduced healthcare costs. ‘Liquid biopsies' that rely upon chemistry for the detection of blood-based endogenous cancer biomarkers such as proteins, circulating tumor DNA or RNA show only limited sensitivity for certain early stage cancers where the tumor mass shedding those biomarkers is smaller. Rather than searching for elusive natural biomarkers shed into the bloodstream, we are employing a promising alternate approach rooted in biology to usurp the highly dysregulated transcriptional pathways that give rise to malignancies to force cancer cells to produce easily detectable ‘synthetic' biomarkers that do not naturally occur. EARLI-001 is a DNA nanoplasmid comprised of a cancer-activated promoter to drive expression of Secreted Embryonic Alkaline Phosphatase (SEAP). The survivin promoter was initially selected for proof of concept because the corresponding anti-apoptotic gene product is widely overexpressed in many cancers, including melanoma, liver, breast, lung, colon and ovarian cancer, while not being expressed in normal adult tissues. The synthetic biomarker SEAP is a secreted variant of human placental alkaline phosphatase, which is only expressed during fetal development and thus has near-zero background in adult blood. EARLI-001 is formulated using a non-viral linear polyethyleneimine and administered intravenously to enable broad tissue distribution and transient transfection of multiple tissues. The sensitivity and specificity of EARLI-001 for tumor detection were established using a variety of cell-derived xenograft models. A single administration of EARLI-001 in immunocompromised mice bearing a modest burden of lung metastases drove cancer-activated SEAP expression that was at least 100-fold higher than the corresponding expression in non-tumor bearing animals. In an immune-competent syngeneic model, EARLI-001 produced 10-fold higher SEAP expression in mice bearing lung metastases of 4T1 mammary tumor cells than naïve mice. EARLI-001 could also discriminate mice bearing orthotopic Hep3B liver xenograft tumors from naïve control mice. Finally, the sensitivity of EARLI-001 in detecting small tumors was demonstrated in a longitudinal study, in which robust cancer-specific SEAP expression occurred even when the lung tumor burden was too low to result in significant changes in organ weight. The toxicology, pharmacokinetics, and biodistribution of EARLI-001 were evaluated in several GLP studies in mice as well as canines, enabling translation to a first-in-man clinical study. This Phase 1 dose escalation study will evaluate the safety, tolerability and PK/PD of EARLI-001 in subjects with locally advanced or metastatic lung cancer and enable further clinical development of the cancer-activated synthetic biomarker platform. Citation Format: Badriprasad Ananthanarayanan, Regina Nieu, Evan Bishop, Shireen Rudina, Alex Harwig, Bijee George, David Suhy. Preclinical development of EARLI-001, a genetic platform producing cancer-activated synthetic biomarkers for the early detection of malignancies [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 2548.

IMMU-24. TEMOZOLOMIDE INDUCED IMMUNE DYSFUNCTION REQUIRES THE PRESENCE OF INTRACRANIAL TUMORS

Abstract Temozolomide was recently shown to cause peripheral and intra-tumoral T cell dysfunction in a dosing schedule dependent fashion. Standard dose (SD) temozolomide (TMZ) resulted in T cell dysfunction precluding response to immune checkpoint inhibition that was avoided with a metronomic dosing (MD) schedule. Building on these studies, we investigated the TMZ-induced immune changes in tumor and non-tumor bearing models to understand the interaction of an intracranial tumor on host immunity. C57BL/6 mice underwent intracranial implantation of GL-261 tumor cells. Tumor bearing animals and naïve animals with no tumor were treated with standard dose (50 mg/kg x 5 days) or metronomic dose (25mg/kg x 10 days) of TMZ. Peripheral blood and spleens were collected for flow cytometry, ELISA and luciferase killing assay. Tumor bearing animals treated with SD TMZ demonstrated an increase in circulating myeloid derived suppressor cells (MDSCs), an upregulation of exhaustion markers on endogenous host CD8 T cells (TIM3, LAG3) and a decrease in IFN-gamma secretion from adoptively transferred T cells (tested via ELISA). The cell killing capability of adoptively transferred T cells was not reduced after exposure to a TMZ treated host. Non-tumor bearing animals treated with SD TMZ did not demonstrate an increase in circulating MDSCs or exhaustion markers on endogenous T cells. IFN-gamma secretion from adoptively transferred T cells was still reduced in these animals. The host immune dysfunction induced by TMZ is dependent on the presence of an intracranial malignancy. The mechanisms causing these changes are under active investigation.

TMIC-42. LOCAL TISSUE METABOLOMICS BASED BIOMARKERS OF RESPONSE TO THERAPY FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a common deadly malignant brain cancer of the central nervous system (CNS), with a median survival of 12–15 months. Scientific advancements are lacking in developing effective therapies for both primary GBM, as well as secondary GBMs, that typically originate as malignant transformation of lower-grade isocitrate dehydrogenase (IDH) 1-mutant tumors. The unique metabolomic profile of IDH1-mutant tumors may present opportunities to develop biomarker signatures of therapeutic efficacy. Microdialysis is an extracellular fluid sampling collection technique utilizing a perfused semipermeable catheter to permit diffusion of molecules between brain interstitium and the perfusate. We hypothesized that microdialysis may identify a metabolomics-based biomarker response to therapy in IDH1-mutant tumors. To test this hypothesis, orthotopic xenografts were generated from two patient-derived GBM lines harboring mutations in IDH1. Perfusates were collected from intra-cranial tumors in aythmic nude mice sampled at baseline and 72h post treatment with temozolomide, an oral alkylating agent used to treat IDH1-mutant gliomas, compared with vehicle treatment, and TMZ-treated non-tumor bearing animals. Perfusates were analyzed via unsupervised metabolomic profiling using both gas and liquid chromatography-mass spectrometry (GC/LC-MS). Tumor specific metabolites such as carnitine and pyruvic acid were detected in microdialysate from tumor bearing mice compared to non-tumor bearing mice. Microdialysis is a feasible technology to identify metabolomics-based biomarker in IDH1-mutant PDX. This work is complemented by parallel analysis of non-IDH1-mutant and TMZ resistant xenografts to yield predictive in vivo tissue biomarkers of drug responsiveness translatable to clinical practice.

Abstract 4553: Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow

Abstract Systemic immunosuppression in cancer is a mechanism of immune escape allowing for cancer progression to metastasis and remains a major pathophysiologic barrier to treatment including immunotherapies. The mechanism of this phenomenon remains relatively obscured. Although immune suppressive humoral factors secreted by primary tumors are thought to be a major cause of immune escape, a major question remains whether factors critical for cancer metastasis also dually regulate systemic immunosuppression to prepare peripheral sites for the eventual encounter with metastasizing cancer cells. To that end, we report here that the master cancer epithelial-mesenchymal transition (EMT) initiating factor Snail1, whose expression is restricted to the primary tumor where it promotes the generation of invasive breast tumor-initiating cells, yet also has a direct role in creating a pro-tumor systemic immune environment to promote metastasis. These dual roles for Snail1 in breast cancer progression has not been elucidated. Using both spontaneous genetic murine model of breast cancer and a syngeneic orthotopic breast cancer model we demonstrate that Snail1 is uniquely required for metastatic spread of breast tumor cells while also directly modulates the bone marrow niche, skewing it towards a suppressive immune phenotype with an upregulation of myeloid derived suppressor cells (MDSCs) and a decrease in antigen presenting cell (APC) populations. Importantly, deletion of Snail1 specifically in primary tumors severely abrogated metastasis and simultaneously restored bone marrow immune populations to levels in non-tumor bearing animals. In vitro co-culture assays demonstrated that humoral factors from Snail1-repleted cancer cells promoted MDSC formation and suppressed APCs when compared to Snail1-deficient cancer cells. Mechanistically, Snail1 modulates systemic immunosuppression specifically through 1) upregulating the expression of GM-CSF, CXCL2, and CCL2, factors important for MDSC induction and recruitment, within the primary tumor to saturate the periphery with these factors; and 2) generating metastatic cells that may suppress immune functions within the metastatic niche. Taken together, these data demonstrate Snail1’s central role in metastatic progression in breast cancer through its direct generation of metastatic cells and suppressing the systemic immune system to increase the chance that these metastatic cells may survive the periphery. Note: This abstract was not presented at the meeting. Citation Format: Mathew Sebastian, Dongjiang Chen, Son Le, Duy Nguyen, Changwang Deng, Dan Jin, Nagheme Thomas, David Tran. Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4553.

Abstract B85: Plasmatic levels of arginine, its precursors and acylcarnitines affected by melatonin at specific time of the day

Abstract The circadian rhythm regulates almost the whole metabolism modulating the concentration of various circulating substances through the clear and dark variation. Melatonin is the main mediator of this bidimensional flow, although studies have shown that metabolic diseases like cancer have already been associated to the imbalance of this rhythm. Actually, melatonin has several anticancer effects extensively described in the literature and proved by our group. Nowadays, metabolomics analysis has been used in cancer science to provide a comprehensive profile of the metabolic changes seeking to uncover characteristics about the tumor behavior. Studies in this field have discovered a lot of new biochemical pathways involved in the homeostasis and disorders. It is noticeable that important tumor features are regulated by essential molecules involved in bioenergy and cell structure, likewise amino acids and fatty acids. Therefore, the comprehension of specific metabolites that may be modulated by the exogenous melatonin is essential to verify its use as cancer adjuvant treatment. The aim of this study was to evaluate the metabolic profile in breast cancer xenograft model under treatment with melatonin across the circadian variation. We used 160 female Balb/c nude athymic mice separated into four groups: tumor/melatonin, tumor/vehicle, nontumor/melatonin, and nontumor/vehicle. The tumor model was developed by implantation of 3 x 106 triple-negative human breast cancer cells (MDA-MB-231) in the mammary fat pad. All the animals received 100 ul of solution by intraperitoneal injection (IP) for five days a week during 21 days. Treated groups received 40 mg/kg of melatonin and nontreated group only received vehicle solution. Blood sample were collected every 3 hours during 24 hours and the plasma was extracted for liquid chromatography and mass spectrometry (LC-MS) analysis using the Absolute IDQ p180 Kit (BioCrates). Our Orthogonal Partial Least Squares (OPLS) analysis showed evident separation of the metabolic profile not only because of the circadian cycle, but also because of the melatonin treatment. By looking for specific pathways we found that tumor/vehicle group showed significant increases in the plasmatic levels of arginine, citrulline, and ornithine, specifically at 6 am and 9 am (p < 0.001). Arginine metabolism is well known to be strongly involved in the initial phase of tumor growth (Lind, 2004; HU et al., 2016), favoring the synthesis of molecules very important for various biologic processes of normal and tumor cells. Beyond the aminoacids we also found significantly raised levels of medium- and long-chain acylcarnitines, specifically at 3 pm (p < 0.001). Acylcarnitines are mainly responsible for the fatty acid metabolism, which is a fundamental energy source. Synthesis of fatty acids is required for membrane synthesis and therefore for cell growth and proliferation. Our results show that tumor-bearing animals treated with melatonin have shown reduced plasma levels of these metabolites, achieving the same levels of non-tumor bearing animals. In summary, this work reveals a strong relationship between the metabolic profile and the entire circadian variation as well as the fact that exogenous melatonin can regulate the metabolites' concentration. This preclinical trial showed increased plasmatic levels of arginine, its precursors and acylcarnitines at specific time of the day, and the treatment with melatonin is able to decrease the serum levels of these metabolites. Thus our results corroborate the literature which characterizes these metabolites as cancer biomarkers and increasingly allows the use of melatonin as an adjuvant therapy for breast cancer. Citation Format: Rubens Paula Junior, Nathália Martins Sonehara, Debora A. P. C. Zuccari. Plasmatic levels of arginine, its precursors and acylcarnitines affected by melatonin at specific time of the day [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B85.

P121: Morphological and functional changes in the population of alveolar macrophages of mice with induced carcinogenesis in the lung

Background Alveolar macrophages (AM) are pulmonary residents of the bone marrow-derived mononuclear phagocyte system that play a critical role in several diverse lung functions and in lung host defense. The role of AM in lung cancer is multifaceted. AM secretion of proinflammatory cytokines has been found to enhance antitumor functions, cytostasis and cytotoxicity. AM have also demonstrated to have protumor functions resulting in tumor progression. Materials and methods 24-week-old male A/f mice were kept in plastic cages under standard laboratory conditions and received food and water ad libitum. Animals were randomly assigned to the following experimental groups: I – control, II – radiation only, III – sulphur dioxide only, IV – sulphur dioxide + radiation. Animals were subjected to total irradiation at dose 1.0 Gy (dose density of 16.6 cGy/min, using a 60Co gamma equipment). After irradiation mice were inhaled air with sulphur dioxide (20 mg/m 3 for 1 h) in separate exposure chamber. At the 20th week of experiment the 20 mice per each group were subjected to autopsy. Excised lungs were fixed in neutral buffered 10% formalin. To obtain an index of tumor incidence, the percentage of tumor – bearing mice per total number of mice in each group was calculated. Tumor multiplicity was defined as the average number of tumors per mice, obtained by dividing the total number of tumors by the total number of mice per group, including non-tumor bearing animals. Bronchoalveolar lavage (BAL) of lungs was performed on 6 mice (animals were anesthetized by an ip injection of sodium thiopental) from each study group at the 1, 7, 15 and 30 days after irradiation and SO2 inhalation. Total cells of BAL were counted using hemocytometer chamber and an optical microscope with a 400× zoom. BAL differential cell (macrophages, lymphocytes, eosinophiles and neutrophils) counts were performed on slides (Romanowsky stain). Phagocytic activity of AM was counted on slides. AM morphometric study was done by measuring the area of each cell (square of cell) and the area of nuclear of each cell (square of nuclear) with use of the original image analyzer computer system. Results Results of the combined effects of radiation and inhalation of sulfur dioxide showed the growth of tumor quantity in the lung: the frequency of adenomas/mouse was more than 42% higher than that of control group. Synergism coefficient was 1.1 in this case. Irradiation at a dose of 1.0 Gy modifies the reaction of free cells of lung on the action of sulfur dioxide, which is manifested in the change of the number of cells in the BAL fluid, the phagocytic activity of the AM, as well as of the changes the morphometric parameters of their cells. Conclusion Thus, if the amount of radiation exposure of BAL cells was significantly reduced by the 1st and 7th days, and after inhalation of sulfur dioxide – tended to increase, then the combined action on the 7th day it was almost half the theoretical (hypothetical) the sum of the separate effects of the studied factors. Exposure to sulfur dioxide significantly slowed the activation of cell count. It was found, that after combined action of the investigated factors the morphological characteristics of macrophages (reducing the square of cells and their nuclei) have changed (7th and 15th day). The absorption activity of phagocytes was significantly reduced (7 day).

Abstract 2143: Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models.

Abstract Anthracyclines (doxorubicin) are one of the most effective and widely used chemotherapeutic agent for the treatment of several cancer types, including early stage and metastatic breast cancer. However, doxorubicin has been known to cause long-term bone damage and growth defects in patients with childhood cancers, which was also confirmed by several preclinical studies. This raises concern regarding the usage of doxorubicin for the treatment of patients with metastatic breast cancer who are already at increased risk of osteolytic bone damage leading to severe bone loss due to. A number of clinical and preclinical studies have indicated that doxorubicin causes both long term and short term bone damages by lowering bone mineral density, increasing bone loss and may disrupt bone homeostasis by directly affecting bone marrow stem cells, however, the underlying mechanism is poorly understood. We have previously reported that treatment with doxorubicin increases circulating levels of TGFβ, a cytokine which has been implicated in the increase in osteoclast differentiation and survival, suppression of osteoblast differentiation and has been associated with advancement of breast cancer to bone metastasis. We hypothesize, breast cancer patients undergoing doxorubicin treatment will experience accelerated bone loss due to an increase in serum TGFβ levels and blocking TGFβ will rescue this bone loss, at least partially. We have tested the efficacy of anti-TGFβ antibody 1D11, in blocking doxorubicin-mediated bone damage in cardiac injection and orthotopic models of breast cancer to bone metastasis. X-ray image analysis revealed that doxorubicin treatment resulted in an eightfold increase in osteolytic lesion areas (P=0.002) and microCT analysis has shown almost 50% decrease in BV/TV (P=0.0005), in a preclinical murine model metastatic breast cancer. Doxorubicin treatment has also decreased BV/TV in non-tumor bearing animals, suggesting a direct damaging effect of this agent to bone at the sites which are not affected by cancer infestation. As anticipated, treatment with anti-TGFβ antibody 1D11 was able to rescue doxorubicin-mediated bone loss in all three preclinical models tested. At the cellular level, direct effect of doxorubicin treatment on the bone marrow microenvironment included decreased osteoblast survival and reduced mineralized matrix formation and increased osteoclast formation, thereby affecting both bone formation and bone resorption. Taken together, we conclude that, doxorubicin therapy decreases bone formation and increases bone resorption causing additional bone loss in patients with osteolytic bone disease and an anti-TGFβ therapy may be affective in controlling that bone loss. Citation Format: Tapasi Rana, Anwesa Chakrabarti, Michael Freeman, Swati Biswas. Anti-TGFβ antibody treatment rescues doxorubicin- mediated bone loss in preclinical osteolytic breast cancer metastasis models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2143. doi:10.1158/1538-7445.AM2013-2143

Abstract 5386: Immunotherapy of bladder cancer using bacillus calmette guérin (BCG): Generation of cytotoxic T-cells is BCG strain dependent

Abstract Introduction: Forty years ago Zbar and colleagues demonstrated that bacillus calmette guérin (BCG) is capable of provoking an immune response against experimentally induced tumors. The conditions for successful tumor immunity were defined by a i) limited number of tumor cells in ii) close contact with a iii) sufficient number of bacteria and iv) the ability of the organism to mount a BCG specific immune response. These findings led to the development of what is currently the standard of care for the treatment of patients with non-muscle invasive bladder cancer. Whether the genetic drift between BCG strains that are routinely used in treatment protocols for bladder cancer affects the generation of successful tumor immunity is unknown. Here we compared two of the commonly used BCG strains (Connaught and Tice) in the treatment of bladder cancer in an animal model. Methods: Naïve or bladder cancer bearing (orthotopic implantation of syngeneic MB49 cells) Bl/6 mice received either a single s.c. injection or weekly intravesical instillation of 107 CFUs of BCG Tice or Connaught. Twelve days after the 4th intravesical treatment or after the single s.c. immunization, the bladders, spleens and draining lymph nodes (DLN) were removed, enzymatically digested and the resulting cell suspensions analyzed by flow cytometry, including assessment of BCG specific CD8+ T-cells. Lymph node extracts were cultivated for the enumeration of BCG CFUs. Tumor bearing animals were assessed for their overall survival. Results: In non-tumor bearing animals, BCG Connaught lead to a more robust influx of adaptive immune cells into the bladder and primed significantly more BCG specific cytotoxic CD8+ T-cells. Compared to BCG Connaught, BCG Tice was less capable to disseminate into the draining lymph nodes (DLN), and induced less Th1 polarized CD4+ T-cells in the bladder, DLN and spleens, as assed by intracellular T-bet staining. These findings were independent of BCG preparation (clinical grade versus laboratory grade) and were seen for identical treatment doses. Finally, mice, orthotopically challenged with MB49 bladder cancer cells, showed significantly better survival if treated with BCG Connaught as compared to BCG Tice. Conclusion: Here we show that different BCG strains significantly impact the immunological response of adaptive immunity. Along with a stronger Th1 immune response, favoring the generation of cytotoxic T-cells, BCG Connaught improves survival as compared to BCG Tice in vivo. These findings implicate potential consequences for clinical practice and corroborate testing of different BCG strains in prospective clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5386. doi:1538-7445.AM2012-5386

Natural Killer Cell Adoptive Immunotherapy Is Hampered by A Rapid Development of NK Cell Dysfunction Characterized by Loss of Effector Mechanisms and Downregulation of the Transcription Factors T-Bet and Eomesodermin; Definition and Possible Mechanism of NK Cell Exhaustion

Abstract 1000Natural killer (NK) cells exhibit in vitro cytotoxicity against many tumor cell types and have an important role in controlling tumor growth, as depletion of NK cells from tumor-bearing mice hastens tumor growth and impairs survival. These data, in combination with results from clinical trials of haploidentical killer immunoglobulin-like receptor (KIR)-ligand mismatched bone marrow transplantation, led to interest in the use of adoptive NK immunotherapy for the treatment of malignancy. Recent clinical results have shown that allogeneic NK cells can be safely administered after chemotherapy and/or irradiation but have also demonstrated limited persistence of the infused NK cells without clear evidence of efficacy. We traced the fate of adoptively infused NK cells in order to delineate the barriers to successful NK immunotherapy using several NK-sensitive murine tumor models.Mice bearing established lymphoma or leukemia received an intravenous infusion of 0.5–1.0×106 (approximately 2.5–5×107/kg body weight) NK cells from allogeneic or syngeneic donors after or concurrently with total body irradiation and bone marrow rescue. Using luciferase +ve NK cells, we first showed that in animals bearing subcutaneous tumors, NK cells homed to lymphoid organs in the first week, followed by progressive localization to and accumulation within the tumor site (Figure 1). In contrast, in non-tumor bearing animals NK cells homed to lymph nodes, spleen and liver with maximal proliferation at the end of the second week. These observations indicated that NK cells fail to eradicate the tumor despite prior demonstration of in vitro sensitivity, successful homing and local accumulation. As expected from these findings, survival was not prolonged by the NK cell infusion. Reisolation of donor NK cells within 18 hours of transfer showed enhanced cytotoxicity (14% vs 2%, p = 0.004) and IFNγ production (48% vs 22%, p = 0.04) compared with naive resting NK cells. In contrast, NK cells isolated from tumor-bearing mice at later time-points beginning d+5 showed loss of IFNγ production (48% early vs 3% late, p = 0.01), decreased expression of the activating receptor NKG2D, and impaired cytotoxicity in chromium release assays. These observations did not relate to over-stimulation through NKG2D, as NK cells from NKG2D−/− animals were also susceptible to acquired dysfunction (although their baseline cytotoxicity was lower than WT NK toward A20 lymphoma). [Display omitted] Eomesodermin and T-bet are transcription factors with important roles in effector functions of CD8+ T cells and NK cells. In T cells T-bet downregulation has been shown to correlate with exhaustion (Kao et al, Nat Imm 2011). Flow cytometry of reisolated NK cells revealed downregulation of Eomesodermin (naive splenic control, d+1 reisolated and d+10 reisolated cells showing 85%, 96%, and 29% expression, respectively) and T-bet (naive splenic control, d+1 reisolated and d+10 reisolated cells showing 82%, 99%, and 59% expression, respectively), correlating with loss of IFNγ production.The phenotype described herein was most dramatic within the tumor and within mice carrying high tumor burdens, but was also present in NK cells reisolated from non-tumor bearing animals that received NK cells, suggesting that homeostatic proliferation after transfer of mature NK cells could also contribute to exhaustion. CFSElo proliferated NK cells showed the most dramatic loss of effector function (chromium release = 42% in unproliferated vs 18% in proliferated cells, p = 0.03) and transcription factor expression (Eomesodermin positive 83% in unproliferated cells vs 18% in proliferated cells, p = 0.002).Collectively, our results suggest that the success of NK cell immunotherapy is limited by an acquired dysfunction that occurs within days after homeostatic proliferation and target encounter and that may be related to the downregulation of transcription factors required for NK effector function. These findings illuminate a previously unappreciated phenomenon and explain why short-term in vitro killing assays have limited utility in predicting the in vivo behaviour of transferred NK cells. Hence, these findings suggest that transferred NK cells become dysfunctional in vivo and that novel approaches may be required in order to circumvent the described dysfunction phenotype. Disclosures:No relevant conflicts of interest to declare.

Toxicity‐reducing potential of extracorporeal affinity adsorption treatment in combination with the auristatin‐conjugated monoclonal antibody BR96 in a syngeneic rat tumor model

Antibody-drug conjugates, comprising monoclonal antibodies (MoAbs) that bind to tumor-associated antigens, display different toxicity profiles compared with radiolabeled MoAbs. Dose-limiting toxicities may include damage to the liver and myelotoxicity. The drug component is the antimitotic agent auristatin, which is 100-1000 times more potent than doxorubicin. Consequently, auristatin antibody-drug conjugates require a high selectivity in tumor targeting to display pronounced activity at well-tolerated doses. We have evaluated the possibility of increasing the therapeutic index of BR96-auristatin by combining the administration of conjugates with subsequent extracorporeal affinity adsorption treatment. Rats were injected with biotinylated, monomethyl auristatin F (MMAF)-conjugated monoclonal antibody BR96. The conjugate was then removed from the circulation by extracorporeal affinity adsorption treatment, 24 hours postinjection using an avidin affinity column. By analyzing blood parameters for 100 days, myelotoxicity, hepatotoxicity, and nephrotoxicity were assessed. Body weight, general status, and tumor size were also recorded. The toxicity-reducing effect of extracorporeal affinity adsorption treatment was evaluated. Extracorporeal affinity adsorption treatment removed 85%-90% of BR96-MMAF from the circulation. Early toxicity-related death was seen in nontumor-bearing animals that were given MMAF-conjugated BR96, in contrast to animals that were given a higher amount of BR96-MMAF with subsequent extracorporeal affinity adsorption treatment, in which all survived 100 days postinjection. Extracorporeal affinity adsorption treatment reduced the loss of body weight, myelotoxicity, and hepatotoxicity. Extracorporeal affinity adsorption treatment can be used to reduce the toxicity associated with administration of BR96-MMAF conjugates, making it possible to increase the amount of conjugates administered. The combined treatment will be further optimized in future studies.

Direct Evidence of Mesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments Using In Vivo Bioluminescent Imaging

Multipotent mesenchymal stromal/stem cells (MSC) have shown potential clinical utility. However, previous assessments of MSC behavior in recipients have relied on visual detection in host tissue following sacrifice, failing to monitor in vivo MSC dispersion in a single animal and limiting the number of variables that can be observed concurrently. In this study, we used noninvasive, in vivo bioluminescent imaging to determine conditions under which MSC selectively engraft in sites of inflammation. MSC modified to express firefly luciferase (ffLuc-MSC) were injected into healthy mice or mice bearing inflammatory insults, and MSC localization was followed with bioluminescent imaging. The inflammatory insults investigated included cutaneous needle-stick and surgical incision wounds, as well as xenogeneic and syngeneic tumors. We also compared tumor models in which MSC were i.v. or i.p. delivered. Our results demonstrate that ffLuc-expressing human MSC (hMSC) systemically delivered to nontumor-bearing animals initially reside in the lungs, then egress to the liver and spleen, and decrease in signal over time. However, hMSC in wounded mice engraft and remain detectable only at injured sites. Similarly, in syngeneic and xenogeneic breast carcinoma-bearing mice, bioluminescent detection of systemically delivered MSC revealed persistent, specific colocalization with sites of tumor development. This pattern of tropism was also observed in an ovarian tumor model in which MSC were i.p. injected. In this study, we identified conditions under which MSC tropism and selective engraftment in sites of inflammation can be monitored by bioluminescent imaging over time. Importantly, these consistent findings were independent of tumor type, immunocompetence, and route of MSC delivery.

Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin–cyclophosphamide chemotherapy

Abnormal accumulation of myeloid-derived suppressor cells (MDSC) is an important mechanism of tumor immune evasion. Cyclophosphamide (CTX) has also been shown in non-tumor bearing animals to cause transient surges in MDSC. Knowledge of MDSC is primarily based on preclinical work, and to date only few published studies have involved cancer patients. The goal of this study was to test the hypothesis that circulating MDSC levels correlate with clinical cancer stage, CTX-based chemotherapy, and metastatic tumor burden. Whole blood was collected from 106 newly diagnosed solid tumor patients (stages I-IV). Percentages of circulating MDSC (Lin(-/Lo), HLA DR-, CD33(+)CD11b(+)) were determined prior to initiation of systemic therapy. In 17 early stage breast cancer patients receiving doxorubicin-cyclophosphamide chemotherapy every 14 days (ddAC) blood was collected on day 1 of each cycle. Circulating MDSC were significantly increased in cancer patients of all stages relative to healthy volunteers. A significant correlation between circulating MDSC and clinical cancer stage was also observed. Moreover, among stage IV patients, those with extensive metastatic tumor burden had the highest percent and absolute number of MDSC. Significant increases in circulating MDSC were observed with ddAC when compared with pretreatment levels. Circulating MDSC levels correlate with clinical cancer stage, ddAC, and metastatic tumor burden. This information must be incorporated into the design of future trials exploring immune-based therapeutic strategies. Pharmacologic modulation of MDSC should also be tested in future clinical trials.

The Expression Signatures of Mammary Tumor Associated Macrophages.

It is well established that an increased density of tumor-associated macrophages (TAMs) correlates with poor prognosis in many types of solid tumors. This evidence is particularly strong for breast cancers. A causal relationship between TAMs and poor prognosis was suggested by experiments whereby a genetic depletion of macrophages in a mouse model of breast cancer caused by the mammary restricted expression of the Polyoma Middle T oncoprotein (PyMT) slowed tumor progression and inhibited metastasis. Subsequent studies in these primary mammary tumors showed that TAMs directly or indirectly promote tumor angiogenesis as well as tumor cell migration, invasion and intravasion. TAMs are also thought to affect the inflammatory context of the tumor microenvironment by suppressing adaptive immune responses that would normally reject the growing tumor. The varied tasks ascribed to TAMs suggested that the tumor microenvironment educates different population of macrophages to perform specific tasks. In this study, we isolated TAMs from the PyMT primary tumors in order to evaluate their gene expression signatures compared to a resident splenic population in order to define specific tumor associated functions. To perform these studies mice that express enhanced green fluorescent protein (eGFP) from the colony stimulating factor 1 receptor (CSF-1R) mononuclear phagocytic restricted promoter were crossed to the PyMT animals to generate offspring with eGFP+ TAMs. Animals were injected with dye-conjugated dextran two hours prior to sacrifice in order to identify phagocytic cells, a characteristic of macrophages. We have established that these eGFP+/dextran+ cells are F4/80+ and define the TAM population. EGFP+/dextran+ TAMs were isolated using flow cytometry from late-stage tumors and splenic macrophages were sorted from non-tumor bearing animals using an identical protocol. These two populations were analyzed on gene expression oligoarrays to better elucidate specific mediators of TAM pathogenicity. We have identified approximately 100 genes whose transcript abundance are up or down regulated in the TAM population including genes mediating angiogenesis, adhesion and inflammation. Furthermore, genes previously described to define the tumor associated suppressor macrophage (MIF-1, MIP1α and TGFβ, high; IL-18, low) were similarly regulated amongst the three biological repeats. To further define individual TAM populations, we used an in vivo invasion assay to isolate a subset of TAMs that promote carcinoma cell motility in vivo. This assay involves the collection of invasive tumor cells and co-migrating invasive TAMs into EGF-containing microneedles placed directly into the primary tumor of an MMTV-PyMT animal. Previously, this assay was used to describe a paracrine loop in which carcinoma cells secrete CSF-1 that binds CSF-1R on TAMs leading to TAM secretion of epidermal growth factor (EGF) that binds the EGF receptor on carcinoma cells and stimulate their motility. Disrupting this paracrine loop is known to block the invasion of both cell types. Invasive TAMs isolated via this assay and separated from invasive carcinoma cells using CD11b magnetic beads were compared by gene expression arrays to TAMs sorted by flow cytometry (F4/80+/dextran+). The transcript abundance of about 200 genes were differentially regulated between these two populations. Together, these two studies illustrate key genes expressed in TAMs that may regulate tumor progression and furthermore, define a specific sub-population of TAMs that directly promotes tumor cell migration and invasion.

The complexities of an apparently simple lung tumor model: The A/J mouse

A simple animal model of tobacco smoke carcinogenesis works as follows: Strain A/J mice are exposed for 5 months to tobacco smoke. They are then given a 4-month recovery period in air before being killed. Lung surface tumors are counted and lung tumor multiplicity (average number of tumors per lung, including non-tumor bearing animals) is calculated. Results obtained in four different laboratories during the past 8 years have consistently shown significant increases in lung tumor multiplicities in tobacco smoke exposed animals. While inhaling to tobacco smoke, strain A mice (but not some other strains) fail to gain weight and immediately after smoke exposure only have about 75% of control weight; however, when removed into air, they regain weight rapidly up to control levels. The counting of surface tumors only may occasionally underestimate total number of lung tumors and thus yield false negatives. At the end of the experiment, the mice are 1-year old and about 80% of the tumors are adenomas, the remainder adenomas with carcinomatous foci or adenocarcinomas. Tobacco smoke does not increase the percentage of adenocarcinomas. Studies with filtered tobacco smoke have suggested that benzo(a)pyrene or tobacco smoke-specific nitrosamines cannot account for lung carcinogenesis in mice; the most likely single agent to cause lung tumors is 1,3-butadiene. A major disadvantage of the assay is its low statistical power. While it is easy to detect a 70–100% decrease in lung tumor multiplicity caused by a chemopreventive agent using group sizes of 20–30 animals, the detection of smaller reductions (20–50%) would require group sizes in the hundreds. From all available evidence it must be concluded that the complex mixture of tobacco smoke, a known human carcinogen, is a rather weak rodent carcinogen.

A Novel Interleukin-IL-6 (IL-6) Dependent Syngeneic Multiple Myeloma (MM) Model in SCID Mice.

IL-6 is a multifunctional cytokine that is implicated in the pathophysiology of several malignant diseases including MM, an incurable malignant plasma cell disorder. IL-6 is known to enhance proliferation, differentiation and survival of malignant plasma cells in MM through an autocrine and/or a paracrine mechanism that involves the inhibition of apoptosis of the malignant cells, induction of resistance to chemotherapy and contribution to angiogenesis. Moreover, elevated levels of IL-6 in serum of patients with MM correlate with disease activity, unfavorable prognosis and refractoriness to standard therapy. Blocking IL-6 has been postulated to be an effective therapy (Klein et al, 1995) and several studies have investigated the effect of blocking IL-6 on MM cells and cell lines both in vitro and in vivo. However, the lack of a reliable IL-6 dependent MM model has hindered these efforts. Recently, mouse plasmacytomas were described as appropriate models to study the biology of human MM (Iankov et al., Immunobiology 2004; 208(5)). The current study describes a new in vivo IL-6 dependent mouse plasmacytoma model in SCID mice. Mice were inoculated subcutaneously with 1 x 106 7TD1 cells, an IL-6 dependent mouse hybridoma/plasmacytoma cell line. Three days after tumor inoculation, mice were treated 2x/week i.p. with either PBS or 25 mg/kg of anti-mouse IL-6 (R & D systems, Clone MP520F3) or control mAb. Thirteen days after tumor implantation the mean tumor volume in the control mAb group and PBS group was 3204 +/− 360 SEM mm3, n = 10; and 2430 +/− 189 SEM mm3, n = 10, respectively. The mean tumor volume in the anti-IL-6 treated group was 635 +/− 149 SEM mm3, n = 10. Serum was tested by ELISA for levels of IL-6 and IgM (a mAb that is produced by 7TD1 cell line). IL-6 serum level was undetectable in naïve non-tumor bearing SCID mice. The IL-6 levels in the PBS treated group and control mAb group were 121 +/− 32 pg/ml and 125 +/− 14 pg/ml, respectively. IL-6 levels in animals treated with rat-anti- mouse IL-6 were not detected due to interference of the mAb with the ELISA. Serum IgM levels in optical density (OD) were 0.02 +/− 0.005 in the naïve non-tumor bearing animals, 0.80 +/− 0.02 in the PBS group, 0.77 +/− 0.03, in the control mAb group, and 0.19 +/− 0.17 in the rat anti-mouse IL-6 group. In conclusion the current study showed that 7TD1cells could be grown in SCID mice. Serum levels of both IgM and IL-6 were significantly elevated in the PBS and control mAb treated tumor-bearing animals. There was a significant reduction in the IgM levels in the rat anti-mouse IL-6 treated group (P <0.0001), a positive correlation between final tumor weight and serum IgM level (P < 0.0001, r2 = 0.782) and a 74% inhibition of tumor growth relative to either control mAb or vehicle control (P <0.0001). Taken together the current study introduces a new IL-6 dependant mouse plasmacytoma model that can be used to study the biology of MM and to test the efficacy of IL-6 blocking agents in vivo.

Antimetastatic effectiveness of amifostine therapy following surgical removal of Sa-NH tumors in mice

The effects of dose per fraction on the ability of amifostine exposure to elevate angiostatin levels in the serum of mice and to inhibit spontaneous metastases formation using the well-characterized murine Sa-NH sarcoma were investigated. Amifostine was administered intraperitoneally at doses of 50, 100, or 200 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8 mm in diameter. Amifostine was again administered immediately following surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Nontumor-bearing control animals were treated using the same dosing and surgery schedules. The average number of pulmonary metastases per animal was determined for each experimental group. A significant reduction ( P < .05) in the average number of pulmonary metastases was observed only in the group of animals exposed to a dose per fraction of 50 mg/kg. A dose of 100 mg/kg was less effective while 200 mg/kg had no effect on metastases formation in this study. The effects of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin were also determined using Western analysis. Correlating with the antimetastatic effect measured, exposure of animals to 50 mg/kg of amifostine resulted in a four-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in both tumor-bearing as well as nontumor-bearing animals. In contrast, a dose of 200-mg/kg amifostine administered intraperitoneally under these conditions had no measurable effect on angiostatin serum levels in this animal system. The enhanced ability of relatively low doses of amifostine to inhibit spontaneous metastases formation suggests that effective antimetastatic therapies with amifostine can be designed with minimal toxic side effects. While the dose responses for angiostatin production and metastases inhibition by amifostine are well correlated, the precise mechanism of action underlying these phenomena is unclear but is suggestive of a redox driven process(es). Semin Oncol 29 (suppl 19):22-28. Copyright 2002, Elsevier Science (USA). All rights reserved.

Inhibition of spontaneous metastases formation by amifostine.

Amifostine was investigated for its ability to inhibit spontaneous metastases formation using the well-characterized murine sarcoma, Sa-NH. Amifostine was administered intraperitoneally at a dose of 50 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8-8.5 mm in diameter. Amifostine was again administered immediately after surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Twenty-one days later, animals were evaluated for the presence of spontaneously developed pulmonary metastases. Nontumor-bearing control animals were sham treated using the same dosing and surgery schedules. Treatment with amifostine appeared to slightly delay tumor growth, that is, 13 vs. 12 days for tumors to reach an average diameter of 8 mm. Amifostine reduced both the incidence of pulmonary metastases formed in experimental animals from 77% to 57% (p < 0.05), and their average number per animal from 12.8 +/- 5.4 (SEM) to 2.9 +/- 1.1 (SEM). The effect of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin was also determined using Western blot analysis. Consistent with the antimetastatic effect, exposure of animals to 50 mg/kg of amifostine resulted in a 4-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in tumor-bearing and nontumor-bearing animals. The effects of amifostine on matrix metalloproteinase (MMP) enzymatic activity was also determined using gelatin zymography. Conditioned growth medium collected from Sa-NH cells grown to confluency was exposed to various concentrations of SH, i.e., 2-[(aminopropyl)amino]ethane-thiol (WR-1065), the active thiol form of amifostine, for either 30 min or 18 hr. WR-1065, as a function of increasing dose and time, inhibited the enzymatic activities of MMP-2 and MMP-9. At a concentration and time of exposure likely to be achieved in vivo, that is, 40 microM and 30 min, MMP-2 and MMP-9 activities were reduced to between 30% and 40% of control values. Consistent with these affects, WR-1065 was also found to be effective in inhibiting the ability of Sa-NH cells to migrate through Matrigel membranes. After an 18-hr exposure under in vitro conditions, WR-1065 at concentrations of 4, 40 and 400 microM, and 4 mM, inhibited Sa-NH migration to 11%, 44%, 81% and 97% of control values, respectively. The abilities of amifostine and its active thiol WR-1065 to stimulate angiostatin production in mice, and to inhibit the MMP enzymatic activities and invasion ability of Sa-NH cells under in vitro conditions, are consistent with the observed antimetastatic effects exhibited against Sa-NH tumors growing in vivo.

Disposition of irinotecan and SN-38 following oral and intravenous irinotecan dosing in mice.

The present study was conducted to quantitate the disposition of irinotecan lactone and its active metabolite SN-38 lactone in mice following oral and intravenous administration, and to evaluate the systemic exposure of irinotecan lactone and SN-38 lactone associated with antitumor doses of irinotecan lactone in mice bearing human tumor xenografts. Nontumor-bearing mice were given a single oral or intravenous irinotecan dose (5, 10, 40, or 75 mg/kg), and serial plasma samples were subsequently obtained. Irinotecan and SN-38 lactone plasma concentrations were measured using an isocratic HPLC assay with fluorescence detection. The disposition of intravenous irinotecan lactone was modeled using a two-compartment pharmacokinetic model, and the disposition of oral irinotecan and SN-38 lactone was modeled with noncompartmental methods. Irinotecan lactone showed biphasic plasma disposition following intravenous dosing with a terminal half-life ranging between 1.1 to 3 h. Irinotecan lactone disposition was linear at lower doses (5 and 10 mg/kg), but at 40 mg/kg irinotecan lactone clearance decreased and a nonlinear increase in irinotecan lactone AUC was observed. The steady-state volume of distribution ranged from 19.1 to 48.1 l/m2. After oral dosing, peak irinotecan and SN-38 lactone concentrations occurred within 1 h, and the irinotecan lactone bioavailability was 0.12 at 10 mg/kg and 0.21 at 40 mg/kg. The percent unbound SN-38 lactone in murine plasma at 1000 ng/ml was 3.4 +/- 0.67%, whereas at 100 ng/ml the percent unbound was 1.18 +/- 0.14%. Irinotecan and SN-38 lactone AUCs in micebearing human neuroblastoma xenografts were greater than in nontumor-bearing animals. Systemic exposure to unbound SN-38 lactone in nontumor-bearing animals after a single oral irinotecan dose of 40, 10, and 5 mg/kg was 28.3, 8.6, and 2.9 ng h/ml, respectively. Data from the present study provide important information for the design of phase I studies of oral irinotecan.