Tumor-induced Changes Research Articles

Detecting Corticospinal Tract Impairment in Tumor Patients With Fiber Density and Tensor-Based Metrics.

Tumors infiltrating the motor system lead to significant disability, often caused by corticospinal tract injury. The delineation of the healthy-pathological white matter (WM) interface area, for which diffusion magnetic resonance imaging (dMRI) has shown promising potential, may improve treatment outcome. However, up to 90% of white matter (WM) voxels include multiple fiber populations, which cannot be correctly described with traditional metrics such as fractional anisotropy (FA) or apparent diffusion coefficient (ADC). Here, we used a novel fixel-based along-tract analysis consisting of constrained spherical deconvolution (CSD)-based probabilistic tractography and fixel-based apparent fiber density (FD), capable of identifying fiber orientation specific microstructural metrics. We addressed this novel methodology’s capability to detect corticospinal tract impairment. We measured and compared tractogram-related FD and traditional microstructural metrics bihemispherically in 65 patients with WHO grade III and IV gliomas infiltrating the motor system. The cortical tractogram seeds were based on motor maps derived by transcranial magnetic stimulation. We extracted 100 equally distributed cross-sections along each streamline of corticospinal tract (CST) for along-tract statistical analysis. Cross-sections were then analyzed to detect differences between healthy and pathological hemispheres. All metrics showed significant differences between healthy and pathologic hemispheres over the entire tract and between peritumoral segments. Peritumoral values were lower for FA and FD, but higher for ADC within the entire cohort. FD was more specific to tumor-induced changes in CST than ADC or FA, whereas ADC and FA showed higher sensitivity. The bihemispheric along-tract analysis provides an approach to detect subject-specific structural changes in healthy and pathological WM. In the current clinical dataset, the more complex FD metrics did not outperform FA and ADC in terms of describing corticospinal tract impairment.

Adipogenesis in Different Body Depots and Tumor Development.

Adipose tissue (AT) forms depots at different anatomical locations throughout the body, being in subcutaneous and visceral regions, as well as the bone marrow. These ATs differ in the adipocyte functional profile, their insulin sensitivity, adipokines’ production, lipolysis, and response to pathologic conditions. Despite the recent advances in lineage tracing, which have demonstrated that individual adipose depots are composed of adipocytes derived from distinct progenitor populations, the cellular and molecular dissection of the adipose clonogenic stem cell niche is still a great challenge. Additional complexity in AT regulation is associated with tumor-induced changes that affect adipocyte phenotype. As an integrative unit of cell differentiation, AT microenvironment regulates various phenotype outcomes of differentiating adipogenic lineages, which consequently may contribute to the neoplastic phenotype manifestations. Particularly interesting is the capacity of AT to impose and support the aberrant potency of stem cells that accompanies tumor development. In this review, we summarize the current findings on the communication between adipocytes and their progenitors with tumor cells, pointing out to the co-existence of healthy and neoplastic stem cell niches developed during tumor evolution. We also discuss tumor-induced adaptations in mature adipocytes and the involvement of alternative differentiation programs.

Abstract 5026: Efficacy of anti-PD-1, IDO inhibitor, chemotherapy and bone-targeting agent on tumor growth in a syngeneic bone metastasis model of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive cancer with treatment options limited to standard-of-care chemotherapy. TNBC patients develop metastases in high incidence, and almost all patients have bone metastases at end-stage disease. Bone metastases are mainly osteolytic, causing severe bone loss that can be prevented by bone-targeting agents. Currently, there is no efficient cure for bone metastases. Activation of the patient’s own immune system by immunotherapies is widely studied in various cancers, but their efficacy on bone metastases is not well established. In this study, we aimed to evaluate the efficacy of immunotherapies in comparison to standard-of-care compounds in a preclinical TNBC bone metastasis model. Mouse 4T1-GFP TNBC cells were inoculated intracardially to immunocompetent female Balb/c mice to model bone metastasis. Treatment of single agents of chemotherapy (cyclophosphamide, 100 mg/kg), bone-targeting agent (zoledronic acid, 0.1 mg/kg), and immunotherapies programmed cell death 1 antibody (mouse anti-PD-1, 10 mg/kg) and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor (epacadostat, 100 mg/kg), and the combination of anti-PD-1 and epacadostat were started on the day following the cancer cell inoculations. The study was terminated about 2 weeks after the inoculations. Tumor burden was evaluated at sacrifice by GFP imaging in skeleton and soft tissues, and tumor-induced bone loss by X-ray imaging. Tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemical stainings of CD3+, CD4+ and CD8+ T cells. Cyclophosphamide decreased skeletal tumor burden and the area of tumor-induced osteolytic bone lesions. It also decreased the number of soft tissue metastases in ovaries and adrenal glands. Zoledronic acid decreased osteolytic lesion area but had no effect on tumor burden. Anti-PD-1, epacadostat and their combination had no effect on tumor burden, on the development of osteolytic lesions, or on the number of soft tissue metastases. Low number or no TILs were observed in the tumors growing in bone obtained from vehicle-treated mice. Immunotherapies did not prevent tumor growth in bone or tumor-induced bone changes in the syngeneic TNBC model. This is probably a consequence of the low number of TILs in the tumors growing in bone. Further studies are needed to evaluate the effects of treatment combinations and to increase the responsiveness to immunotherapies especially in bone metastases. Citation Format: Tiina Emilia Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jussi M. Halleen, Jenni Bernoulli. Efficacy of anti-PD-1, IDO inhibitor, chemotherapy and bone-targeting agent on tumor growth in a syngeneic bone metastasis model of triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5026.

Abstract 1634: Orthotopic and bone metastasis prostate cancer models using the 22Rv1 cell line

Abstract Prostate cancer is the most common cancer in men. Upon diagnosed malignancy the growth of the tumors is androgen driven in a majority of the cases. For that reason, the tumors are treated with androgen deprivation therapy (ADT) to stop or reduce tumor growth. Later, the tumors often become resistant to ADT and at this stage the disease is described as castration-resistant prostate cancer (CRPC). Bone metastasis is one of the hallmark of disease progression. Metastatic disease can be either androgen sensitive or castration resistant (mCRPC) in which the mortality rate is over 50%. Prostate cancer metastases are commonly formed in bones. The 22Rv1 human prostate carcinoma cell line is androgen sensitive and shown to express androgen receptor (AR). The 22Rv1 cells are an invaluable model system to study AR function, the efficacy of existing drugs and to design novel anti-AR therapies that also target non-truncated regions of AR. The aim of this study was to establish predictive orthotopic and intratibial preclinical in vivo prostate cancer models that can be used in drug development when targeting cancer cells and their local environment. Male athymic nude mice (5-6 weeks old) and NOG mice (over 20 weeks old) were used in the study. 2.5 × 105 22Rv1 cells were inoculated orthotopically into the prostate of NOG mice, and 0.5 × 106 cells were inoculated intratibially into the bone marrow cavity of athymic nude mice. 22Rv1 human prostate cancer cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% iFBS, 2 mM L-Glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/l D-Glucose and P/S. Tumor growth was followed by PSA measurements using Human PSA ELISA assay. In addition, in the intratibial model, tumor-induced bone changes were monitored by X-ray imaging of the hind limbs. Prostates and hind limbs were collected at sacrifice, fixed in 10% NBF, and processed to paraffin blocks for further histological analysis. Sections of prostate were stained with hematoxylin-eosin (HE) and AR. Tumor-bearing and contralateral (healthy) tibias were stained with HE - OrangeG, MGT, TRAP, and AR. For enhanced visualization of tumor models in 3D, we reconstructed the tissue from serial sections using a tailored computational pipeline based on applying a deformable model for corresponding points from adjacent sections. The observed tumor take rate was 100% in both models. Confirmed by IHC, tumors formed in both the orthotopic and intratibial models expressed AR. 22Rv1 cells formed osteoblastic - lytic mixed bone lesions in the intratibial model. Based on lesion areas, randomization of the tumor bearing mice in the intratibial model can be done after 2 weeks from cancer cell inoculation. In conclusion, both presented models are suited to test the anticancer efficacy of new drug candidates. In addition, the intratibial model can be used to test the efficacy of novel drug candidates on prostate cancer cells in bone metastatic microenvironment. Citation Format: Justyna Zdrojewska, Tiina E. Kähkönen, Jenni H. Mäki-Jouppila, Masi Valkonen, Pekka Ruusuvuori, Jussi M. Halleen, Jenni Bernoulli. Orthotopic and bone metastasis prostate cancer models using the 22Rv1 cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1634.

Age-related changes in T lymphocytes of patients with head and neck squamous cell carcinoma

IntroductionThe number of aging cancer patients has increased continuously and will do so further in the future. The immune system of elderly people experiences critical changes over the time. Therefore, tumor-induced changes in the immune system are believed to differ in young and elderly cancer patients as well.MethodsThe effect of aging on the immune system was measured in peripheral blood lymphocytes (PBL) of healthy volunteers (n = 48, 21–84 yrs.) divided into three different age groups. Seventy years was set as a cut-off for defining subjects as elderly. Results were compared to two groups of adult cancer patients, which donated PBL and tumor infiltrating lymphocytes (TIL): young cancer patients (40–69 yrs.; blood: n = 13; TIL: n = 17) and elderly cancer patients (70–90 yrs.; blood: n = 20; TIL: n = 15) with head and neck squamous cell carcinoma (HNSCC). Frequencies and phenotypes of CD4+ and CD8+ T cells as well as regulatory T cells (Treg) were assessed by flow cytometry.ResultsWe observed lower frequencies of CD8+ cytotoxic T cells during aging in both groups. Frequencies of tumor infiltrating regulatory T cells were significantly higher than in the peripheral blood but showed a significant decline in older tumor patients. With increasing age, expression of immunosuppressive CD73 and CCR7 was lower and expression of PD1 elevated on peripheral T cells in healthy volunteers and tumor patients.ConclusionImmunosenescence takes place in healthy donors and cancer patients. Our results suggest that in elderly tumor patients, the immune system is impaired and the tumor-induced immune escape is less pronounced. The increased expression of PD1 implies the potential for effective immunotherapies in elderly, as treatment with checkpoint inhibitors could be more beneficial for elderly HNSCC patients.

Breast cancer induces systemic immune changes on cytokine signaling in peripheral blood monocytes and lymphocytes.

BackgroundIt is increasingly recognized that cancer progression induces systemic immune changes in the host. Alterations in number and function of immune cells have been identified in cancer patients’ peripheral blood and lymphoid organs. Recently, we found dysregulated cytokine signaling in peripheral blood T cells from breast cancer (BC) patients, even those with localized disease.MethodsWe used phosphoflow cytometry to determine the clinical significance of cytokine signaling responsiveness in peripheral blood monocytes from non-metastatic BC patients at diagnosis. We also examined the correlation between cytokine signaling in peripheral monocytes and the number of tumor-infiltrating macrophages in paired breast tumors.FindingsOur results show that cytokine (IFNγ) signaling may also be dysregulated in peripheral blood monocytes at diagnosis, specifically in BC patients who later relapsed. Some patients exhibited concurrent cytokine signaling defects in monocytes and lymphocytes at diagnosis, which predict the risk of future relapse in two independent cohorts of BC patients. Moreover, IFNγ signaling negatively correlates with expression of CSF1R on monocytes, thus modulating their ability to infiltrate into tumors.InterpretationOur results demonstrate that tumor-induced systemic immune changes are evident in peripheral blood immune cells for both myeloid and lymphoid lineages, and point to cytokine signaling responsiveness as important biomarkers to evaluate the overall immune status of BC patients.FundingThis study was supported by the Department of Defense Breast Cancer Research Program (BCRP), The V Foundation, Stand Up to Cancer (SU2C), and Breast Cancer Research Foundation (BCRF).

Abstract LB-A01: Establishment of a novel prostate cancer bone metastasis model in humanized mice and early efficacy results of pembrolizumab

Abstract Background: Programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1) expression is typically low in primary prostate cancer but increases at advanced stages. However, targeting PD-1 or PD-L1 has not proven efficacy in prostate cancer patients, and recent clinical trials are directed to combination therapies. About 85% of advanced prostate cancer patients develop skeletal metastases, and the focus in this context should be addressed to the treatment of these metastases. These efforts have been hindered by the lack of relevant preclinical bone metastasis models in immunocompetent mice. We aimed to establish a prostate cancer bone metastasis model in humanized mice and to assess pembrolizumab efficacy in the established model. Materials and methods: Two million LNCaP human prostate cancer cells were inoculated into tibia bone marrow of male CIEA NOG® mice engrafted with human CD34+ hematopoietic stem cells to generate humanized mice. Serum prostate-specific antigen (PSA) levels were measured at 4 weeks, and the mice were allocated to receive either pembrolizumab (anti-PD-1, Keytruda®, 5 mg/kg, Q5D) or human IgG4 isotype control for 6 weeks. Tumor growth was monitored by measuring serum PSA levels. Tumor-induced bone changes were monitored by measuring serum levels of the bone formation marker N-terminal propeptide of type I procollagen (PINP), and by X-ray imaging of tibia. Changes in quantity of circulating T cells was monitored by flow cytometry. At study termination, tissue samples were collected for histological and immunohistochemical evaluation of tumor-infiltrating lymphocytes (TILs). Results: A tumor take of 90% was observed in the humanized mice evaluated by serum PSA levels at endpoint. No significant differences were observed in serum PSA levels, but in a few mice treated with pembrolizumab, only modest tumor growth was observed. Intratibial LNCaP tumors induced osteoblastic-mixed lesions as evaluated by X-ray imaging. No differences were observed in the bone lesion area or in serum PINP levels between the isotype control and pembrolizumab groups. No changes in circulating levels of CD4+, CD8+, or CD4+CD8+ cells were observed. Histology will be performed to confirm tumor morphology and overall tumor viability, and immunohistochemical analysis will reveal the quantity and location of TILs in the tumor. Conclusions: A novel preclinical model of bone metastasis in humanized mice was established. Intratibial prostate cancer tumors induced osteoblastic mixed bone lesions and increased serum PSA levels, mimicking the clinical situation in patients. Resembling recent clinical findings, no or modest responses with pembrolizumab as monotherapy were observed. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Azusa Tanaka, Philip Dube, Jussi M Halleen, Jenni Bernoulli. Establishment of a novel prostate cancer bone metastasis model in humanized mice and early efficacy results of pembrolizumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A01. doi:10.1158/1535-7163.TARG-19-LB-A01

Abstract B144: Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer with treatment options limited to standard-of-care chemotherapy. TNBC patients develop metastases in high incidence, and almost all patients have bone metastases at end-stage disease. Bone metastases are mainly osteolytic, causing severe bone loss that can be prevented by bone-targeted agents. Currently, there is no efficient cure for bone metastases. Activation of the patient’s own immune system by immunotherapies is widely studied in various cancers, but their efficacy on bone metastases is not well established. In this study, we aimed to evaluate the efficacy of immunotherapies in comparison to standard-of-care compounds in a preclinical TNBC bone metastasis model. Materials and methods: Mouse 4T1-GFP TNBC cells were inoculated intracardially to immunocompetent female Balb/c mice to model bone metastasis. Treatment of single agents of chemotherapy (cyclophosphamide, 100 mg/kg), bone-targeting agent (zoledronic acid, 0.1 mg/kg), and immunotherapies programmed cell death 1 antibody (mouse anti-PD-1, 10 mg/kg) and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor (epacadostat, 100 mg/kg), and the combination of anti-PD-1 and epacadostat were started on the day following the cancer cell inoculations. The study was terminated 12-13 days after the inoculations. Tumor burden was evaluated by GFP imaging and tumor-induced bone loss by X-ray imaging at sacrifice. Tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemical stainings of CD3+, CD4+ and CD8+ T cells. Results: Cyclophosphamide decreased skeletal tumor burden and the area of tumor-induced osteolytic bone lesions. Zoledronic acid decreased the area of osteolytic lesions but had no effect on skeletal tumor burden. Anti-PD-1, epacadostat and their combination had no effect on skeletal tumor burden or in the development of osteolytic lesions. Low number or no TILs were observed in the tumors growing in bone obtained from vehicle-treated mice. Conclusions: Immunotherapies did not prevent tumor growth in bone or tumor-induced bone changes in the syngeneic TNBC model. This is probably caused by the low number of TILs in the tumors growing in bone. Further studies are needed to evaluate the effects of treatment combinations and to increase the responsiveness to immunotherapies especially in bone metastases. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Jussi M Halleen, Jenni Bernoulli. Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B144. doi:10.1158/1535-7163.TARG-19-B144

Abstract 2814: Systemic and local bone metastasis models for immuno-oncology drug development

Abstract Bone metastases are common and count 30-70% of metastases in breast, lung, and bladder cancer, and 80% of multiple myeloma patients have bone disease. Despite recent progress in cancer treatment, bone metastases remain incurable. Novel immunotherapies have the potential to cure also bone metastatic disease. Here we have established and validated syngeneic models with a focus on bone metastasis that could be used in preclinical efficacy studies. Syngeneic models were established for breast (4T1-GFP), multiple myeloma (5TGM1), bladder (MBT-2) and lung cancer (KLN-205). The cells were inoculated into systemic circulation (4T1 intracardially and 5TGM1 into tail vein) or bone marrow (MBT-2 and KLN-205). In the 4T1 model, tumor burden was assessed by ex vivo GFP imaging and in the 5TGM1 model by measuring serum IgG2b paraprotein levels during the study. Tumor-induced bone changes were followed by X-ray imaging in all models. Hind limbs were analyzed by histology and immunohistochemistry for tumor-infiltrating lymphocytes (TILs). The effects of standard-of-care compounds were assessed in the 4T1 (cyclophosphamide, 100 mg/kg or zoledronic acid, 0.1 mg/kg) and 5TGM1 (bortezomib, 1 mg/kg) models. The effect of anti-PD-1 treatment (200 µg/dose) was evaluated in the MBT-2 model. In the 4T1 model, osteolytic bone lesions formed within 13 days. In addition to bone metastases, about 50% of the mice had metastases in lungs, ovaries, kidneys and adrenal glands based on GFP imaging. Cyclophosphamide decreased the tumor burden and the area of osteolytic bone lesions. Zoledronic acid decreased the osteolytic lesion area but had no effect on tumor burden. Low number or no TILs were observed. In the 5TGM1 model, osteolytic lesions were observed and the study was ended at day 35. Metastases in ovaries, kidneys and adrenal glands were observed in about 30% of the mice. Bortezomib decreased serum paraprotein compared to vehicle treated mice. In both models, cachexia and paraplegia were occasionally observed. Moderate number of CD3+ TILs were observed in the tumors. In the intratibial MBT-2 and KLN-205 models, large osteolytic lesions were observed within 25 days, and in the KLN-205 model also lung metastases were observed. Anti-PD-1 treatment decreased osteolytic tumor area in the MBT-2 model. Moderate number of CD3+ and low number of CD4+ and CD8+ TILs were observed in the tumors growing in bone and also in the lung metastases. A high incidence of bone metastases was observed in all models. The use of systemic models allows studying the effects of test compounds in prevention or treatment of metastases. Intratibial models can be used when the primary interest is in tumor growth in bone microenvironment. Mimicking the clinical situation, none of the SOC compounds could prevent tumor growth completely, and therefore combination therapies are warranted for better overall efficacy. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jussi M. Halleen, Arne Scholz, Jenni Bernoulli. Systemic and local bone metastasis models for immuno-oncology drug development [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 2814.

Cancer and cancer survival modulates brain and behavior in a time-of-day-dependent manner in mice

Improvements in breast cancer therapy/diagnosis have substantially increased the cancer survivor population, although many survivors report persistent mental health issues including fatigue, mood and anxiety disorders, and cognitive impairments. These behavioral symptoms impair quality-of-life and are often associated with increased inflammation. Nocturnal rodent models of cancer are critical to the identification of the neurobiological mechanisms underlying these behavioral changes. Although both behavior and immunity display distinct diurnal patterns, most rodent research in this field is performed during the rodents’ inactive (light) period, which could potentially undermine the conclusions and clinical relevance. Therefore, here we tested the extent to which mammary tumors or tumor resection (“survivors”) in mice affects behavior and neuroinflammation in a nyctohemeral (day versus night)-dependent manner. Indeed, only the dark (active) phase unmasked fatigue-like behavior and altered novel object investigation for both tumor-bearing and -resected mice relative to surgical controls. Several inflammatory markers were expressed in a time-of-day-dependent manner (lower in the dark phase) in the blood and brains of surgical control mice, whereas this temporal pattern was absent (IL-1β, CXCL1, Myd88, Cd4) or reversed (C3) in the respective tissues of tumor-bearing and -resected mice. Taken together, these data indicate that the time of day of assessment significantly modulates various persistent and transient tumor-induced behavioral and immune changes.

Dovitinib dilactic acid reduces tumor growth and tumor-induced bone changes in an experimental breast cancer bone growth model

Advanced breast cancer has a high incidence of bone metastases. In bone, breast cancer cells induce osteolytic or mixed bone lesions by inducing an imbalance in bone formation and resorption. Activated fibroblast growth factor receptors (FGFRs) are important in regulation of tumor growth and bone remodeling. In this study we used FGFR1 and FGFR2 gene amplifications containing human MFM223 breast cancer cells in an experimental xenograft model of breast cancer bone growth using intratibial inoculation technique. This model mimics bone metastases in breast cancer patients. The effects of an FGFR inhibitor, dovitinib dilactic acid (TKI258) on tumor growth and tumor-induced bone changes were evaluated. Cancer-induced bone lesions were smaller in dovitinib-treated mice as evaluated by X-ray imaging. Peripheral quantitative computed tomography imaging showed higher total and cortical bone mineral content and cortical bone mineral density in dovitinib-treated mice, suggesting better preserved bone mass. CatWalk gait analysis indicated that dovitinib-treated mice experienced less cancer-induced bone pain in the tumor-bearing leg. A trend towards decreased tumor growth and metabolic activity was observed in dovitinib-treated mice quantified by positron emission tomography imaging with 2-[18F]fluoro-2-deoxy-D-glucose at the endpoint. We conclude that dovitinib treatment decreased tumor burden, cancer-induced changes in bone, and bone pain. The results suggest that targeting FGFRs could be beneficial in breast cancer patients with bone metastases.

Colorectal Cancer and Mitochondrial Dysfunctions of the Adjunct Adipose Tissues: A Case Study.

Excess body weight has been causally linked to an increased risk of different cancer types, including colorectal cancer (CRC) but the mechanisms underlying this association are practically unknown. We investigate redox state-superoxide (SO) generation rate, activity of complex I in electron transport chain (ETC) of mitochondria and of dinitrosyl iron complexes by electron paramagnetic resonance; activity of matrix metalloproteinase (gelatinase) MMP-2 and MMP-9 by gel zymography of adipose tissues (AT) from 46 patients (64.0 ± 1.6 y.o.) with CRC (II–III stages, pT2–3N0–2M0) in the AT adjacent to tumor (ATAT) and at a distance of 3 cm from the tumor (ATD) to follow the connection of the AT redox state with some of the tumor microenvironment indicators. We have incubated the AT species with the tumor necrosis factor α (TNF-α) to follow its influence on the measured values. As a control, normal AT (NAT) obtained during the liposuction is used. Tumor-induced changes in mitochondrial ETC of ATAT, particularly for Complex I, lead to the enhanced SO generation and consequent oxidative modifications of DNA in ATAT (up to 6.1 times higher than that in NAT and 3.7 times higher than that in ATD, p < 0.05). Gelatinase activity in ATAT is significantly higher than in ATD. A considerable effect of TNF-α on ATAT and ATD (but not on NAT, i.e., only on the tissues where the reprogramming of metabolism has already occurred under the influence of tumor) manifested in increase of cellular hypoxia, gelatinase activity, and SO generation rate is observed. The results can be used for better understanding the mechanism(s) of metabolic symbiosis of tumor and AT as well as serving as a basis for new therapeutic approaches.

Language function shows comparable cortical patterns by functional MRI and repetitive nTMS in healthy volunteers.

In preoperative planning, fMRI and repetitive navigated transcranial magnetic stimulation (rTMS) repeatedly revealed differences in the detected language sites, which can be attributed to tumor-induced oxygenation changes impairing the accuracy of fMRI. We therefore compared the accordance of those techniques in healthy subjects using exactly the same tasks in both investigations. 19 healthy right-handed subjects performed object naming, pseudoword reading, verb generation, and action naming during fMRI at 3T and rTMS. For rTMS language mapping, we stimulated 46 cortical spots over the left hemisphere; each site was stimulated for three times. Language positive points during rTMS for one, two, or three errors out the three stimulations per spot (1/3, 2/3, 3/3) were exported via DICOM, and compared to the positive fMRI clusters. As a result of this comparison, the best correlation was observed between 3/3 errors and fMRI for pseudoword reading and verb generation with t-values of pu < 0.001, uncorrected for multiple comparisons, on average across the whole rTMS-spot map. We found a close spatial agreement between several rTMS-spots (2/3 and 3/3 errors) and fMRI clusters accentuated in the frontal lobe, followed by the parietal lobe and less in the temporal lobe. Compared to the fMRI clusters, there was a higher congruence for 2/3 and 3/3 errors than for 1/3 errors. Overall, results of language mapping in healthy subjects by fMRI and rTMS correspond well yet depending on the used language task.

Abstract 3810: Ovarian tumor-induced changes in macrophage phenotype are associated with tumor metastasis

Abstract Background: Ovarian cancer (OVCA) differs from other gynecologic malignancies in its dissemination pattern where the tumor migrates in a diffuse intrapelvic and -abdominal manner. OVCA also triggers a variety of cellular and/or humoral immune responses to a variety of tumor-associated proteins. OVCA is mostly diagnosed at late stages where tumor has already metastasized, indicating an escape from immune responses. Despite several immune response factors, ovarian tumor metastasizing to omentum indicates presence of one or more immune evasion mechanisms. Immune system recognizes and eliminates pathogenic products and tumor cells and macrophages play a critical role in this process. Macrophages in tumor condition express anti-inflammatory cytokines that induce immune suppression. Cellular immune response maximizes killing efficacy of macrophages and proliferation of CD8+ T cells. M2 macrophages are tumor-associated macrophages involved in active growth and progression of tumors. However, the mechanism of ovarian tumor-induced changes in M1 macrophages to M2 phenotype is not well understood. Therefore, the goal of our study was to determine the tumor-induced changes in macrophage phenotype and cytotoxic T cells in association with OVCA metastasis. Materials and Methods: This study was performed in exploratory design using ovarian specimen from normal subjects (60-80 years, n=10) and ovarian malignant tumors at early (n=16, 4 from each histologic type of OVCA) and late stages (n =20) as well as omental tissues from normal (n=3) and OVCA patients (n= 3). Tissue specimen were processed for immunohistochemistry (IHC), Western blotting (WB) and gene expression studies. Expression of M1 and M2 macrophages, CD8 and PI3K was examined by immunohistochemistry (IHC), Western blotting (WB) and qPCR. Results: Compared with normal ovaries, more M1 and M2 macrophages were localized in tumor tissues. However, compared with early-stage OVCA, the frequency of M2 macrophages was significantly higher in ovarian tumors and omental tissues at late OVCA. Intensity of PI3K was also higher in ovarian tumors and omental tissues at late OVCA compared with normal ovarian tissues. Frequency of CD8+T cells was higher in tumor ovarian samples in comparison with normal ovarian tissues. Conclusion: Results of this study suggest that the growth and metastasis of ovarian tumors are associated with increased conversion of M1 macrophages to M2 phenotype. This change in macrophage phenotype may be mediated by the increase in PI3K expression. Support: Swim Across America. Citation Format: Aparna Yellapa, Pincas Bitterman, Sameer Sharma, Sanjib Basu, Animesh Barua. Ovarian tumor-induced changes in macrophage phenotype are associated with tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3810.

Abstract 838: Synergistic in vivo activity of the ATR inhibitor BAY 1895344 in combination with the targeted alpha therapy radium-223 dichloride in a preclinical tumor model mimicking bone metastatic castration-resistant prostate cancer (mCRPC)

Abstract The integrity of the genome in eukaryotic cells is secured by complex signalling pathways, known collectively as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival, or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). The ATR kinase is activated by a broad spectrum of DNA damages, including double-strand breaks (DSBs) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in cancers characterized by increased DNA damage, deficiency in DNA damage repair or replication stress. Radium-223 dichloride (Xofigo®) is the first approved targeted alpha therapy. It is indicated for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceral metastatic disease, based on improvement of overall survival. It is a calcium-mimetics that selectively binds to hydroxyapatite targeting areas of high bone turnover such as bone metastases, thereby exhibiting strong cytotoxic effects on adjacent cells via induction of DNA DSBs. We assessed the anti-tumor efficacy of combination treatment with ATR inhibitor (ATRi) BAY 1895344 and radium-223 dichloride in an intratibially injected prostate cancer model mimicking CRPC with bone metastases. In vivo analyses addressed the optimization of the dosing schedule as well as dose-response of BAY 1895344 in radium-223 combination setting. Levels of intra-tumor DNA damage (P-H2AX) were assessed to demonstrate the proposed mode-of-action. Here, we show that combination treatment with ATRi BAY 1895344 and radium-223 exhibits synergistic anti-tumor activity in the intratibial LNCaP xenograft model of mCRPC, achieving best efficacy when BAY 1895344 is applied 24 hours after treatment with radium-223, as indicated by direct reduction of tumor burden in the bone, lower serum tumor marker (PSA), and smaller areas of tumor-induced changes in bone. With this optimized schedule, a very low dose of BAY 1895344 (3% of single-agent MTD) was effective in combination with radium-223. Our findings strongly suggest that the combination of DNA damage induction by radium-223 with DDR inhibition by ATRi BAY 1895344 creates a potential new treatment option for CRPC patients with bone metastases. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965). Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Arne Scholz, Mari Suominen, Kirstin Meyer, Eleni Lagkadinou, Dominik Mumberg. Synergistic in vivo activity of the ATR inhibitor BAY 1895344 in combination with the targeted alpha therapy radium-223 dichloride in a preclinical tumor model mimicking bone metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 838.

Abstract 709: Anti-PD-1 therapy reduces bone lesion growth in a novel syngeneic bladder cancer bone metastasis model

Abstract Bladder cancer is a common and aggressive type of cancer occurring both in women and in men. At diagnosis, lymph node involvement is frequently observed and in some cases metastatic spread can already be seen at early stage. With advanced stages, the metastatic incidence increases and the most common metastatic sites are bones, lung, liver and peritoneum. Bone metastases count about 50% of all metastases and the 5-year survival rate in metastatic disease is only 15%. Bone metastases are incurable but can be treated to reduce tumor burden in bone, and new therapies such as immunotherapies hold the potential to treat patients with bone metastatic bladder cancer. The aim of the study was to establish a novel syngeneic model for bladder cancer bone metastasis that could be used to study efficacy of new immunotherapies, and to test efficacy of anti-PD-1 therapy in the model. In the model establishment study, an intratibial injection of varying number of murine MBT-2 bladder cancer cells was given to 5-6 weeks old female C3H/HeN mice. Tumor-induced bone changes were followed by X-ray imaging once a week. At sacrifice, tumor-bearing tibias were collected and evaluated by histology. In the efficacy study, 5x105 MBT-2 cells were used. The mice were stratified to treatment groups based on similar bone lesion areas at 10 days after cancer cell inoculations and treated with anti-PD-1 therapy (RMP1-14) or isotype control (rat IgG2a, 200 µg per dose) at Q3D schedule. Bone lesion growth was followed by X-ray imaging one week after start of treatment and at sacrifice after three weeks on treatment. In the model establishment study, tumor take rate of 50% was observed. MBT-2 cells induced an osteolytic bone reaction resulting in substantial bone loss in the mice. The osteolytic bone lesions started to be visible between 7-14 days and the maximum duration of the study was 4 weeks from the cancer cell inoculations. Histology confirmed a large tumor and decreased bone mass in the inoculated tibia. In the efficacy study, treatment with anti-PD-1 reduced bone lesion growth. A trend towards decreased bone lesion area was seen already at one week on treatment and a significant reduction of bone lesion area was seen at sacrifice. A novel syngeneic bladder cancer bone metastasis model was established. According to the observed significant effects on bone lesion growth by anti-PD-1 treatment this model can be used in preclinical efficacy assessment of new immunotherapies as monotherapy or in combination with other therapies. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli, Arne Scholz. Anti-PD-1 therapy reduces bone lesion growth in a novel syngeneic bladder cancer bone metastasis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 709.

A Role for Hypocretin/Orexin in Metabolic and Sleep Abnormalities in a Mouse Model of Non-metastatic Breast Cancer

We investigated relationships among immune, metabolic, and sleep abnormalities in mice with non-metastatic mammary cancer. Tumor-bearing mice displayed interleukin-6 (IL-6)-mediated peripheral inflammation, coincident with altered hepatic glucose processing and sleep. Tumor-bearing mice were hyperphagic, had reduced serum leptin concentrations, and enhanced sensitivity to exogenous ghrelin. We tested whether these phenotypes were driven by inflammation using neutralizing monoclonal antibodies against IL-6; despite the reduction in IL-6 signaling, metabolic and sleep abnormalities persisted. We next investigated neural populations coupling metabolism and sleep, and observed altered activity within lateral-hypothalamic hypocretin/orexin (HO) neurons. We used a dual HO-receptor antagonist to test whether increased HO signaling was causing metabolic abnormalities. This approach rescued metabolic abnormalities and enhanced sleep quality in tumor-bearing mice. Peripheral sympathetic denervation prevented tumor-induced increases in serum glucose. Our results link metabolic and sleep abnormalities via the HO system, and provide evidence that central neuromodulators contribute to tumor-induced changes in metabolism.

Brain tumors disrupt the resting-state connectome.

Brain tumor patients often experience functional deficits that extend beyond the tumor site. While resting-state functional MRI (rsfMRI) has been used to map such functional connectivity changes in brain tumor patients, the interplay between abnormal tumor vasculature and the rsfMRI signal is still not well understood. Therefore, there is an exigent need for new tools to elucidate how the blood‑oxygenation-level-dependent (BOLD) rsfMRI signal is modulated in brain cancer. In this initial study, we explore the utility of a preclinical model for quantifying brain tumor-induced changes on the rsfMRI signal and resting-state brain connectivity. We demonstrate that brain tumors induce brain-wide alterations of resting-state networks that extend to the contralateral hemisphere, accompanied by global attenuation of the rsfMRI signal. Preliminary histology suggests that some of these alterations in brain connectivity may be attributable to tumor-related remodeling of the neurovasculature. Moreover, this work recapitulates clinical rsfMRI findings from brain tumor patients in terms of the effects of tumor size on the neurovascular microenvironment. Collectively, these results lay the foundation of a preclinical platform for exploring the usefulness of rsfMRI as a potential new biomarker in patients with brain cancer.

Cancer Cachexia: Cause, Diagnosis, and Treatment

Patients with cancer frequently experience unintended weight loss due to gastrointestinal (GI) dysfunction caused by the malignancy or treatment of the malignancy. However, others may present with weight loss related to other symptoms not clearly associated with identifiable GI dysfunction such as anorexia and early satiety. Cancer cachexia (CC) is a multifactorial syndrome that is generally characterized by ongoing loss of skeletal muscle mass with or without fat loss, often accompanied by anorexia, weakness, and fatigue. CC is associated with poor tolerance of antitumor treatments, reduced quality of life (QOL), and negative impact on survival. Symptoms associated with CC are thought to be caused in part by tumor-induced changes in host metabolism that result in systemic inflammation and abnormal neurohormonal responses. Unfortunately, there is no single standard treatment for CC. Nutrition consequences of oncologic treatments should be identified early with nutrition screening and assessment. Pharmacologic agents directed at improving appetite and countering metabolic abnormalities that cause inefficient nutrient utilization are currently the foundation for treating CC. Multiple agents have been investigated for their effects on weight, muscle wasting, and QOL. However, few are commercially available for use. Considerations for choosing the most appropriate treatment include effect on appetite, weight, QOL, risk of adverse effects, and cost and availability of the agent.

Abstract 2983: Tumor induced stromal STAT1 deregulates mammary tissue homeostasis and accelerates breast cancer

Abstract The tumor microenvironment (TME) – the dynamic tissue space in which the tumor exists – plays a significant role in tumor initiation and is a key contributor in cancer progression. Little is known about the tumor-induced changes in the adjacent tissue stroma. Herein, we sought to explore tumor-induced changes in the TME at the morphological and molecular level to further characterize cancer progression. We show that tumor-adjacent mammary glands (TAGs) display altered branch morphology, increased expression of αSMA particularly in myofibroblasts, and an increased capacity to form mammospheres in 2D suspension culture. FACS analysis showed that TAGs contain an increased number of Lin-CD24+/CD49+ enriched mammary gland stem cell (MaSCs) population, suggesting deregulated tissue homeostasis in TAGs. We conducted comparative transcriptomics on TAGs and contralateral control glands. Meta-analysis on differentially expressed genes from our RNA-seq dataset plus two breast cancer stromal patient microarray datasets identified shared upregulation of STAT1, which we verified in tumor-adjacent tissues. Knockdown of STAT1 in caveolin-deficient mouse embryonic fibroblasts (CAFs) cocultured with human breast cancer cells altered cancer cell proliferation, further suggesting the role of STAT1 as a stromal contributor of tumorigenesis. Furthermore, in our proof-of-concept in vivo experiment, co-treatment with fludarabine, a FDA-approved STAT1 activation inhibitor and DNA synthesis inhibitor, in combination with doxorubicin, showed enhanced therapeutic efficacy in treating mouse mammary gland tumors. Our results demonstrate that stromal STAT1 expression could promote tumor progression and is a potential therapeutic target for breast cancer. Citation Format: Victoria R. Zellmer, Patricia M. Schnepp, Sarah L. Fracci, Xuejuan Tan, Erin N. Howe, Siyuan Zhang. Tumor induced stromal STAT1 deregulates mammary tissue homeostasis and accelerates breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2983. doi:10.1158/1538-7445.AM2017-2983