Consistent tumorigenesis with self-assembled hydrogels enables high-powered murine cancer studies

Preclinical cancer research is heavily dependent on allograft and xenograft models, but current approaches to tumor inoculation yield inconsistent tumor formation and growth, ultimately wasting valuable resources (e.g., animals, time, and money) and limiting experimental progress. Here we demonstrate a method for tumor inoculation using self-assembled hydrogels to reliably generate tumors with low variance in growth. The observed reduction in model variance enables smaller animal cohorts, improved effect observation and higher powered studies.

A novel treatment for recurrent localized cervical cancer using point-of-care ethyl cellulose ethanol ablation with concurrent cytotoxic therapy.

e17507 Background: Historically, the only curative option for patients with recurrent localized cervical cancer after platinum-based chemotherapy and radiation is pelvic exenteration. For non-surgical candidates, treatment options include systemic therapy with chemotherapy, bevacizumab or pembrolizumab. However, these therapies are not curative. Addition of local ablative therapy to systemic therapy is a promising option, as this combination can induce a more robust local antitumor immune response. We developed a polymer-assisted ethanol ablative therapy, Point-of-care Ethanol Ethyl Cellulose (PEEC), that overcomes the main shortcoming of traditional ethanol ablation: off-target ethanol leakage. We previously demonstrated the success of PEEC in pre-clinical head and neck and breast cancer models. Here, we hypothesized that combination of reduced tumor acidity using sodium bicarbonate (bicarb) and decreased regulatory T cells using systemic or local chemotherapy would prime the tumor immune microenvironment for PEEC as a novel treatment strategy for recurrent localized cervical cancer. Methods: An HPV16 E6/E7+ TC1-Luc cell line was used to establish a syngeneic cervicovaginal tumor model in C57BL/6 mice. First, intratumoral PEEC was compared to sham ablation (saline). Tumor bearing mice were randomized to receive either PEEC or sham (n = 5 each) 2 days after tumor inoculation. Tumor volume and growth were measured with calipers and a Perkin Elmer in vivo imaging system (IVIS) respectively; tumors were imaged on days 1, 2, 3, 7, 14 and 28. Tumor weight was measured by weighing reproductive tracts. Next, we tested whether local and/or systemic cyclophosphamide (cyclo) with bicarb improved response to PEEC. Tumor bearing mice were randomized to receive systemic or local cyclo+bicarb and/or PEEC. The same ablation and tumor monitoring schedule was used, with bicarb (200 mM) added to the drinking water and cyclo (50 mg/kg) given either intraperitoneally or locally on day 1 following tumor inoculation. Results: Tumors treated with PEEC had significantly smaller volumes by day 7 and onward compared to sham (p < 0.005). Tumors treated with PEEC also had significantly decreased tumor weights (at necropsy) compared to sham (p < 0.0002). The PEEC groups showed prolonged survival compared to sham (p < 0.05). Local cyclo+bicarb+PEEC therapy was superior to all other treatment groups with no measurable tumor luminescence signal (via IVIS) in all five mice (5/5) on day 14 and onward. Systemic cyclo+bicarb+PEEC resulted in 4/5 mice with no signal. PEEC alone led to no signal in 3/5 mice. Sham alone showed tumor progression in all 5 mice. Conclusions: PEEC ablation is enhanced by concurrent cytotoxic therapy and significantly controls HPV16 E6/E7+ cervicovaginal tumor growth, supporting future clinical translation for treatment of recurrent localized cervical cancer.

Consistent Tumorigenesis with Self-Assembled Hydrogels Enables High-powered Murine Cancer Studies

Preclinical cancer research is heavily dependent on allograft and xenograft models, but current approaches to tumor inoculation yield inconsistent tumor formation and growth, ultimately wasting valuable resources (e.g., animals, time, and money) and limiting experimental progress. Here we demonstrate a method for tumor inoculation using self-assembled hydrogels to reliably generate tumors with low variance in growth. The observed reduction in model variance enables smaller animal cohorts, improved effect observation and higher powered studies.

Temporal Changes in Immune Responses within the Tumor Microenvironment in the 4T1.2-HER2 Mammary Tumor Model

Background: The murine 4T1.2 triple-negative breast cancer model is widely used, but is poorly immunogenic with no defined tumor-associated antigens. A modified 4T1.2 model has been developed that stably expresses a surrogate tumor antigen, human epidermal growth factor receptor-2 (HER2). The goal of the current study was to characterize host immune responses in the 4T1.2-HER2 tumor model, focusing on the tumor microenvironment (TME) during the early stage of tumor development. Methods: Female BALB/c mice were orthotopically inoculated with 4T1.2-HER2 tumor cells and sacrificed at day (D) 6, 9, 12, 15 and 18 post tumor inoculation. The phenotype and function of tumor-infiltrating immune cells were assessed. Results: 4T1.2 and 4T1.2-HER2 tumor cells had similar proliferation rates in vitro. In contrast to the rapid progression of the parental 4T1.2 model, the 4T1.2-HER2 model demonstrated initial tumor growth followed by spontaneous tumor regression by D18 post tumor inoculation, which was not observed in scid mice. Following tumor regression, mice demonstrated either a second phase of tumor outgrowth or complete tumor rejection. Within the TME, the percentage of T cells was reduced at D9 and increased during tumor regression through D18 (p<0.05), whereas the percentage of myeloid-derived suppressor cells (MDSCs) increased during the initial tumor growth and was reduced by D18 (p<0.01). There was a stepwise increase in the percentage of IFNg+, IL-2+ and perforin+ T cells and NK cells peaking at D12-15. Furthermore, tumor regression occurred concurrently with HER2-specific IFNg production from tumor-infiltrating immune cells at D12 and D15 (p<0.05). During the second phase of 4T1.2-HER2 tumor growth, tumor volume was negatively correlated with immune infiltration (r=0.662, p=0.052). Conclusions: These results suggest that the integration of a surrogate tumor antigen, human HER2, into the clinically relevant, yet poorly immunogenic 4T1.2 breast cancer model enhanced its immunogenicity and induced HER2-specific immune responses.

Effects of Three Consecutive Days of Morphine or Methadone Administration on Analgesia and Open-Field Activity in Mice with Ehrlich Carcinoma

This study assessed the exploratory behavioral responses in BALB/c mice inoculated with Ehrlich ascitic carcinoma after 3consecutive days of treatment with morphine or methadone. Fifty-three female mice, 60 ± 10 d old, were used. Seven days after intraperitoneal tumor inoculation (2 × 106 cells), the animals were randomized into 7 groups: morphine 5 mg/kg (MO5), morphine 7.5 mg/kg (MO7.5), morphine 10 mg/kg (MO10), methadone 2.85 mg/kg (ME2.85), methadone 4.3 mg/kg (ME4.3), methadone5.7 mg/kg (ME5.7), and 0.9% NaCl (Saline) (n = 7). Drug treatments were administered subcutaneously every 6 h for 3 d. The animals were evaluated for analgesia using the mouse grimace scale (MGS) and for general activity using the open field test. The MGS was performed before tumor inoculation (day 0), on day 7 at 40, 90, 150, 240, and 360 min after drug injection, and on days 8 and 9 at 40, 150, 240, and 360 min after drug injection. The open field test was performed before tumor inoculation(day 0), on day 7 after inoculation at 40, 90, 150, 240, and 360 min after drug injection, and on days 8 and 9 after inoculation at 40, 150, and 360 min after drug injection. MGS results indicated that administration of morphine promoted analgesia for up to 240 min. Conversely, methadone reduced MGS scores only at 40 min. All tested doses promoted a significant dose-dependent increase in the total distance traveled and the average speed, and increase that was markedly pronounced on days 8 and 9 as compared with day 7. The frequencies of rearing and self-grooming decreased significantly after morphine or methadone administration. Despite the difference in analgesia, both drugs increased locomotion and reduced the frequency of rearing and self-grooming as compared with the untreated control animals.

Silencing of sinusoidal DDR1 reduces murine liver metastasis by colon carcinoma

Liver metastasis depends on the collagenous microenvironment generated by hepatic sinusoidal cells (SCs). DDR1 is an atypical collagen receptor linked to tumor progression, but whether SCs express DDR1 and its implication in liver metastasis remain unknown. Freshly isolated hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs), that conform the SCs, expressed functional DDR1. HSCs expressed the largest amounts. C26 colon carcinoma secretomes increased DDR1 phosphorylation in HSCs and KCs by collagen I. Inhibition of kinase activity by DDR1-IN-1 or mRNA silencing of DDR1 reduced HSCs secretion of MMP2/9 and chemoattractant and proliferative factors for LSECs and C26 cells. DDR1-IN-1 did not modify MMP2/9 in KCs or LSECs secretomes, but decreased the enhancement of C26 migration and proliferation induced by their secretomes. Gene array showed that DDR1 silencing downregulated HSCs genes for collagens, MMPs, interleukins and chemokines. Silencing of DDR1 before tumor inoculation reduced hepatic C26 metastasis in mice. Silenced livers bore less tumor foci than controls. Metastatic foci in DDR1 silenced mice were smaller and contained an altered stroma with fewer SCs, proliferating cells, collagen and MMPs than foci in control mice. In conclusion, hepatic DDR1 promotes C26 liver metastasis and favors the pro-metastatic response of SCs to the tumor.

P02.05 Investigation of a syngeneic a-PD-1 antibody to treat murine 4T1 mammary carcinoma

BackgroundMany cancers acquire mechanisms to evade immunosurveillance by activating immune checkpoint pathways, which suppress the antitumor immune responses. Monoclonal antibodies (ab’s) targeting immune checkpoints, such as CTLA-4 and PD-1, have shown excellent results in several cancers and are currently being investigated in clinical trials for various malignancies. The clinically tested a-CTLA-4 (Ipilimumab) and a-PD-1 (Nivolumab and Pembrolizumab) ab’s are fully human or humanized ab’s, respectively. However, most studies conducted in mice utilize a xenogeneic a-PD-1 ab originating from rat, IgG2a RMP1-14 clone. This has been proposed to cause adverse effects in the commonly used 4T1 mammary carcinoma model of triple negative breast cancer (TNBC). Repeated administration of xenogeneic a-PD-1 ab’s in this model results in fatal hypersensitivity reactions in tumor bearing mice, and unlike human TNBC, the 4T1 cell line is generally poorly responsive to immune checkpoint inhibitors. Recently, a semi-syngeneic recombinant a-PD-1 ab has been developed by transferring the variable regions of RMP1-14 onto a murine IgG1e3 constant region.Materials and MethodsTesting xenogeneic and semi-syngeneic a-PD-1 ab with and without a-CTLA-4 ab in BALB/c mice carrying 4T1 luciferase positive tumors.ResultsIn this study, we compared a semi-syngeneic recombinant a-PD-1 ab to the original xenogeneic RMP1-14 clone for treatment of luciferase positive 4T1 carcinomas. Surprisingly, the semi-syngeneic a-PD-1 ab was not able to circumvent the fatal hypersensitivity reactions. Still, the combination therapy of a-CTLA-4 and the semi-syngeneic a-PD-1 ab significantly reduced tumor volume in 4T1-luciferase tumor bearing mice compared to isotype control-treated mice already from day 16 post tumor inoculation (day 8 post treatment-initiation). In contrast, xenogeneic a-PD-1/a-CTLA-4 treated mice did not show significant difference from the control group until 24 days post tumor inoculation and never to the same degree. Furthermore, analysis of the T cell responses towards the murine tumor-associated antigen AH-1, revealed that treatment with syngeneic a-PD-1/a-CTLA-4 ab gave a significantly stronger CD8+ T cell response over both control mice and mice treated with xenogeneic a-PD-1/a-CTLA-4 ab.ConclusionsThese studies indicate that the semi-syngeneic a-PD-1 IgG1e3 ab might be a more efficient and translatable a-PD-1 ab for preclinical in vivo studies, which is important for the future investigation of immune checkpoint inhibitor therapy.Disclosure InformationI. Skandorff Pedersen: A. Employment (full or part-time); Significant; InProTher Aps. K. Orfin: A. Employment (full or part-time); Significant; InProTher Aps. K.N. Nielsen: A. Employment (full or part-time); Significant; InProTher Aps. P.J. Holst: A. Employment (full or part-time); Significant; InProTher Aps. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; InProTher Aps.

Positive Effects of Preventive Nutrition Supplement on Anticancer Radiotherapy in Lung Cancer Bearing Mice

(1) Background: Radiotherapy (RT) is one of the major treatments for non-small cell lung cancer, but RT-associated toxicities usually impede its anticancer effect. Nutrient supplementation has been applied for cancer prevention or a complementary measure to anticancer therapy. Here, we explored the influence of total nutrition supplementation before and after cancer occurrence on the anticancer benefit and side effects of RT. (2) Methods: C57BL/6JNarl mice were inoculated with Lewis lung carcinoma cells and then treated with radiotherapy. TNuF, a total nutrition formula, was prescribed by oral gavage. In the preventive groups, TNuF supplementation started from seven days before tumor inoculation. In the complementary groups, TNuF supplementation began after tumor inoculation. (3) Results: TNuF successfully enhanced the anticancer effect of RT against primary tumor and lung metastasis. Additionally, the complementary supplement improved the high serum TNF-α level and the wasting of sartorius muscle in mice receiving RT. In histologic and molecular analysis, TNuF was observed to modulate EGFR, apoptosis, and VEGF and PD-1/PD-L1 pathways. Furthermore, the anticancer benefit of the preventive supplement was comparable to that of the complementary administration. (4) Conclusions: Our results demonstrated that the prescription of the TNuF total nutrition formula before and after cancer diagnosis attains similar benefits in testing subjects with typical anticancer RT. TNuF is also a potential sensitizer to anti-PD-1 immune therapy.

Can Exercise Counteract Cancer Cachexia? A Systematic Literature Review and Meta-Analysis

Background: Cancer-cachexia is associated with chronic inflammation, impaired muscle metabolism and body mass loss, all of which are classical targets of physical exercise. Objectives: This systematic review and meta-analysis aimed to determine the effects of exercise on body and muscle mass in cachectic cancer hosts. Data Sources: PubMed/Medline, EMBASE, CINHAL, ISI Web of Science, and Cochrane Library were searched until July 2019. Study Selection: Trials had to be randomized controlled trials or controlled trials including cancer patients or animal models with cachexia-inducing tumors. Only sole exercise interventions over at least 7 days performed in a controlled environment were included. Data Extraction: Risk of bias was assessed and a random-effects model was used to pool effect sizes by standardized mean differences (SMD). Results: All eligible 20 studies were performed in rodents. Studies prescribed aerobic (n = 15), strength (n = 3) or combined training (n = 2). No statistical differences were observed for body mass and muscle weight of the gastrocnemius, soleus, and tibialis muscles between the exercise and control conditions (SMD = ‒0.05, 95%CI-0.64-0.55, P = 0.87). Exercise duration prior to tumor inoculation was a statistical moderator for changes in body mass under tumor presence ( P = 0.04). Limitations: No human trials were identified. A large study heterogeneity was present, probably due to different exercise modalities and outcome reporting. Conclusion: Exercise does not seem to affect cancer-cachexia in rodents. However, the linear regression revealed that exercise duration prior to tumor inoculation led to reduced cachexia-severity, possibly strengthening the rationale for the use of exercise in cancer patients at cachexia risk.

Precision probiotic therapy enhances immune checkpoint therapy efficacy in melanoma bearing mice.

e14195 Background: Immune checkpoint inhibitor therapy, ICT, achieves remissions in melanoma patients but factors modulating response are not well defined. Our group (Frankel et al. Neoplasia 2017) and others have identified specific gut microbiota associated with improved ICT response. Recently, we identified specific gut microbiota that induce adaptive immune responses and potentiate ICT (Tanoue et al. Nature 2019). In this study, we determined whether this predefined consortia of gut microbiota augment ICT efficacy in melanoma bearing mice. Methods: Mice (C57BL/6, 6-8wk old, female, Jackson, n = 4-12 mice) received ± antibiotic water (penicillin G 1500U/mL + streptomycin 2mg/mL) for 6 d to deplete gut microbiota. Mice were then inoculated with 105 B16F10 melanoma cells SQ. At d 4, 8, 12 post-tumor inoculation, 0.2 mg anti-mCTLA4 + anti-mPD1 antibodies (Bio X Cell) were administered IP. Precision probiotic therapies included Vedanta Bioscience VE800 (Tanoue et al., Nature 2019), VE804 (same as VE800 without R. lactatiformans and F. ulcerans), VE411 (four Clostridial firmicutes) (Narushima, 2014), and Lactobacillus acidophilus (ATCC 4356) probiotics were given via gavage (1x109 cfu) starting day +1 after tumor inoculation and 3xwkly. Loss of survival was defined as death or tumor diameters ≥ 2 cm. Tumor growth inhibition, TGI = (1- mean treated tumor volume/mean control tumor volume) x 100%. Tumor mononuclear cells were isolated for flow cytometry for murine CD4, CD8, and CD11c. Results: TGI in mice with intact gut microbiota and treated with ICT was 84 ± 4% (SEM). Pre-treatment antibiotics reduced TGI to 38 ± 11%. Groups treated with Vedanta VE800, VE804, and VE411 exhibited TGIs of 77 ± 9, 61 ± 8, and 69%, respectively, whereas treatment with Lactobacillus acidophilus achieved TGIs 57%. VE800 treated mice had significantly increased length of survival compared to mice treated with antibiotics (p = 0.0008, log-rank test). Length of survival was not significantly different between groups with intact gut microbiota and those pretreated with antibiotics and dosed with VE800 (p = 0.52, log-rank test). ICT increased tumor CD4 cells to 11% from 2% and CD8 cells to 9% from 1%., however pre-treatment with antibiotics reduced CD4 cells to 4% and CD8 cells to 1%. Conclusions: Defined consortia of gut microbiota facilitate ICT efficacy. These preclinical studies lay the foundation for optimizing the host response to ICT.