565 Combinatorial HSCs and anti-PD-1 therapy in microsatellite stable colorectal cancer

BackgroundColon cancer (CRC) is the second leading cause of cancer-related deaths in the US. CRC incidence is on the rise and there is an alarming increase in young onset CRC cases. Immune checkpoint inhibitors (ICIs) have yielded promising anti-tumor results in microsatellite instable high (MSI-high) patients, which represent only 15% of tumors. The remaining 85% are denoted as microsatellite stable (MSS) and are unresponsive to ICI. Using a murine glioma model, our group has previously found the combination of anti-PD-1 and a transfer of hematopoietic stem cells (HSCs) can sensitize mice that are resistant to anti-PD-1 alone. We evaluated survival after treatment with this combinatorial platform 3 or 5 days post-implantation in subcutaneous CRC-bearing mice and also phenotyped the splenic compartment of mice at endpoint.Methods1x106 MSS CRC cells, CT26, were subcutaneously injected into the right flank of BALB/cJ mice. 3 or 5 days later, HSCs were isolated from naïve BALB/cJ mice and injected through the tail vein into CT26-bearing mice and were also given 10 mg/kg anti-PD-1. Mice were given 3 additional doses of anti-PD-1 for a total of doses either every 3 or 5 days. Mice were sacrificed when tumors reached 1.5 cm at its widest point and spleens were excised and stained for flow cytometry.ResultsWhen mice were treated with HSC/anti-PD-1 3 days post-tumor implantation, we observed a statistically significant increase in survival in mice that received combinatorial HSCs and anti-PD-1 relative to no treatment control mice (p=0.0034, Mantel-Cox long-rank test) as well as mice that received HSCs alone (p=0.0462, Mantel-Cox log-rank test. In the same 3 day cohort, no differences in the frequency of T cell populations were observed. However, we found mice that received this combination therapy had a significant increase in the frequency of splenic CD11c+ MHC II+ dendritic cells (DCs) relative to no treatment control mice (p=0.0364, Mann-Whitney t test). When mice were treated with HSC/anti-PD-1 5 days post-tumor implantation, we found a statistically significant increase in survival of mice treated with combinatorial HSCs and anti-PD-1 compared to no treatment control mice (p=0.0024, Mantel-Cox log-rank test) and relative to mice that received HSC monotherapy (p=0.0462, Mantel-Cox log-rank test).ConclusionsThese results suggest combinatorial HSCs and anti-PD-1 represents a promising therapeutic axis in a murine model of MSS CRC. In addition, the increase in splenic DCs suggests the mechanism behind this anti-tumor response may be expansion of DCs within the periphery.Ethics ApprovalAll animal work approved through University of Florida IACUC # 201910777

Leveraging the Treg-intrinsic CTLA4–PKCη signaling pathway for cancer immunotherapy

BackgroundOur previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These studies have employed adoptive transfer of germline PKCη-deficient (Prkch−/−) Tregs into Prkch+/+ mice prior to tumor implantation. Here, we extended these findings into a biologically and clinically more relevant context.MethodsWe have analyzed the role of PKCη in antitumor immunity and the tumor microenvironment (TME) in intact tumor-bearing mice with Treg-specific or CD8+ T cell-specific Prkch deletion, including in a therapeutic model of combinatorial treatment. In addition to measuring tumor growth, we analyzed the phenotype and functional attributes of tumor-infiltrating immune cells, particularly Tregs and dendritic cells (DCs).ResultsUsing two models of mouse transplantable cancer and a genetically engineered autochthonous hepatocellular carcinoma (HCC) model, we found, first, that mice with Treg-specific Prkch deletion displayed a significantly reduced growth of B16–F10 melanoma and TRAMP-C1 adenocarcinoma tumors. Tumor growth reduction was associated with a less immunosuppressive TME, indicated by increased numbers and function of tumor-infiltrating CD8+ effector T cells and elevated expression of the costimulatory ligand CD86 on intratumoral DCs. In contrast, CD8+ T cell-specific Prkch deletion had no effect on tumor growth or the abundance and functionality of CD8+ effector T cells, consistent with findings that Prkch−/− CD8+ T cells proliferated normally in response to in vitro polyclonal or specific antigen stimulation. Similar beneficial antitumor effects were found in mice with germline or Treg-specific Prkch deletion that were induced to develop an autochthonous HCC. Lastly, using a therapeutic model, we found that monotherapies consisting of Treg-specific Prkch deletion or vaccination with irradiated Fms-like tyrosine kinase 3 ligand (Flt3L)-expressing B16–F10 tumor cells post-tumor implantation significantly delayed tumor growth. This effect was more pronounced in mice receiving a combination of the two immunotherapies.ConclusionThese findings demonstrate the potential utility of PKCη inhibition as a viable clinical approach to treat patients with cancer, especially when combined with adjuvant therapies.

Doxorubicin Paradoxically Ameliorates Tumor-Induced Inflammation in Young Mice

Doxorubicin (DOX) is one of the most widely used chemo-therapeutic agents in pediatric oncology. DOX elicits an inflammatory response in multiple organs, which contributes to DOX-induced adverse effects. Cancer itself causes inflammation leading to multiple pathologic conditions. The current study investigated the inflammatory response to DOX and tumors using an EL4-lymphoma, immunocompetent, juvenile mouse model. Four-week old male C57BL/6N mice were injected subcutaneously with EL4 lymphoma cells (5 × 104 cells/mouse) in the flank region, while tumor-free mice were injected with vehicle. Three days following tumor implantation, both tumor-free and tumor-bearing mice were injected intraperitoneally with either DOX (4 mg/kg/week) or saline for 3 weeks. One week after the last DOX injection, the mice were euthanized and the hearts, livers, kidneys, and serum were harvested. Gene expression and serum concentration of inflammatory markers were quantified using real-time PCR and ELISA, respectively. DOX treatment significantly suppressed tumor growth in tumor-bearing mice and caused significant cardiac atrophy in tumor-free and tumor-bearing mice. EL4 tumors elicited a strong inflammatory response in the heart, liver, and kidney. Strikingly, DOX treatment ameliorated tumor-induced inflammation paradoxical to the effect of DOX in tumor-free mice, demonstrating a widely divergent effect of DOX treatment in tumor-free versus tumor-bearing mice.

The Effects of IFN-γ and IL-4 Expression on PCNA Expression in Transplanted Bone Tumors

Objective: The rabbit VX2 bone tumor model is an ideal experimental animal model for studying malignant bone tumors, but few studies on cytokines and tumor proliferation. This paper is aimed to study the effect of interferon-γ (IFN-γ) and interleukin-4 (IL-4) on the expression of proliferating cell nuclear antigen (PCNA) in tumor tissue. Methods: The experiment included 30 Japanese white rabbits divided into an experimental group (n=15) and a control group (n=15) according to the random number method. Peripheral blood and tumor tissue were collected at 1 and 2 weeks after tumor implantation. We used enzyme-linked immunosorbent assay to detect the expression levels of IFN-γ and IL-4 in peripheral blood and PCNA in tumor tissue. After the rabbits were euthanasia, the tissues were taken for pathological examination. Results: After tumor implantation, the expression level of IFN-γ in the experimental group in the 2nd week was significantly lower than that in the 1st week and 2nd week in the control group (p-value<0.005). In contrast, the expression level of IL-4 in the experimental group in the 2nd week was significantly higher than that in the 1st week and 2nd week in the control group (p-value<0.005). The expression level of PCNA in the experimental group in the 2nd week was significantly higher than that in the 1st week (P<0.005), and the PCNA expression in the 1st and 2nd week in the experimental group was higher than that in the 1st and 2nd week in the control group (p-value<0.005). In the 1st and 2nd week of tumor implantation, the correlation between IFN-γ and PCNA was negatively correlated(r=－0.566, p-value=0.028; r=－0.604, p-value=0.017), while the correlation between IL-4 and PCNA was positively correlated(r=0.583, p-value=0.023; r=0.884, p-value<0.005). Conclusion: In the VX2 bone tumor model of rabbit, with the extension of time after tumor implantation, there was a negative correlation between IFN-γ and PCNA and a positive correlation between IL-4 and PCNA.

Accuracy of an urine-based liquid biopsy genetic test in detecting renal pelvic cancer.

e16500 Background: Renal pelvis cancer is accounting for about two-thirds of upper tract urinary carcinoma. Lesions in renal pelvis often lead to a differential diagnosis that requires ureteroscopic biopsy. Besides causing discomfort and pain, ureteroscopy prior to surgery increases risk of tumor implantation and recurrence. Although urine cytology and FISH were applied, more effective non-invasive diagnostic method is imperative for diagnosis of renal pelvic cancer. Methods: A total of 141 patients with one or more lesions in renal pelvis detected by ultrasound were enrolled. 50-ml first-void urine sample was collection before ureteroscopic biopsy and was analysis by Genetron uro-18 genes panel, by which mutations in 17 genes and methylation status of ONECUT2 were analysis using high-throughput sequencing. If mutations and/or ONECUT2 methylation were detected in urine sediment DNA, the patients were considered as high risk of renal pelvis cancer. The results of the urine-based liquid biopsy genetic test were compared with pathology report and urine FISH results of urinary cells. Results: According to pathological analysis with ureteroscopic biopsy, 42 out of 141 patients (29.7%) were diagnosis with renal pelvic cancer: 13 low-grade, 24 high-grade, 5 equivocal. For 33 renal pelvic cancer patients with FISH testing result of urinary cells, only 54.5% (18/33) samples were positive. By Genetron uro-18 genes test, 95 patients (67.4%) were classified as high risk and 46 (32.6%) patients were low risk. The overall specificity was 88.0% (88/95) and overall sensitivity was 92.9% (39/42), which is significant higher than FISH test of urinary cells. Conclusions: With high specificity and sensitivity, Genetron uro-18 genes test could be incorporated in renal pelvic cancer diagnosis, especially for patients with a ultrasound detectable lesions. Ureteroscopy prior to surgery could be reduced, which may lead to decrease risk of tumor implantation and recurrence.

Over 1000 nm Near-Infrared Multispectral Imaging System for Laparoscopic In Vivo Imaging

In this study, a laparoscopic imaging device and a light source able to select wavelengths by bandpass filters were developed to perform multispectral imaging (MSI) using over 1000 nm near-infrared (OTN-NIR) on regions under a laparoscope. Subsequently, MSI (wavelengths: 1000–1400 nm) was performed using the built device on nine live mice before and after tumor implantation. The normal and tumor pixels captured within the mice were used as teaching data sets, and the tumor-implanted mice data were classified using a neural network applied following a leave-one-out cross-validation procedure. The system provided a specificity of 89.5%, a sensitivity of 53.5%, and an accuracy of 87.8% for subcutaneous tumor discrimination. Aggregated true-positive (TP) pixels were confirmed in all tumor-implanted mice, which indicated that the laparoscopic OTN-NIR MSI could potentially be applied in vivo for classifying target lesions such as cancer in deep tissues.

Depth of tumor implantation affects response to in situ vaccination in a syngeneic murine melanoma model

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically ‘cold’ tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190–230 mm3, they were grouped into a ‘wave’ and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine). Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia. Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors. SC tumors reached the target tumor volume significantly faster compared with ID tumors. Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not. Further, the ‘mobile’ or ‘fixed’ phenotype of tumors predicted response to therapy, regardless of intended implantation depth. These findings were then extended to additional immunotherapy regimens in four separate tumor models. These data indicate that the physical ‘fixed’ versus ‘mobile’ characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment. Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

Activity of tumor-associated macrophage depletion by CSF1R blockade is highly dependent on the tumor model and timing of treatment

Tumor-associated macrophages (TAMs) are abundant in solid tumors where they exhibit immunosuppressive and pro-tumorigenic functions. Inhibition of TAM proliferation and survival through CSF1R blockade has been widely explored as a cancer immunotherapy. To further define mechanisms regulating CSF1R-targeted therapies, we systematically evaluated the effect of anti-CSF1R treatment on tumor growth and tumor microenvironment (TME) inflammation across multiple murine models. Despite substantial macrophage depletion, anti-CSF1R had minimal effects on the anti-tumor immune response in mice bearing established tumors. In contrast, anti-CSF1R treatment concurrent with tumor implantation resulted in more robust tumor growth inhibition and evidence of enhanced anti-tumor immunity. Our findings suggest only minor contributions of CSF1R-dependent TAMs to the inflammatory state of the TME in established tumors, that immune landscape heterogeneity across different tumor models can influence anti-CSF1R activity, and that alternative treatment schedules and/or TAM depletion strategies may be needed to maximize the clinical benefit of this approach.

EXTH-22. THE CNS PENETRATING TAXANE TPI 287 AND THE AURKA INHIBITOR ALISERTIB IMPROVE SURVIVAL IN VIVO

Glioblastoma is the most common primary malignant brain tumor in adults and has a poor prognosis with current standard of care. The AURKA inhibitor alisertib exhibits antiproliferative activity against glioblastoma in vitro including our previous work. Unlike current clinically used taxane drugs, the novel taxane TPI 287 penetrates the CNS. In this study we stereotactically implanted human GB9 xenografts into nude mice brains and tested the activity of alisertib alone, TPI 287 alone and both together compared to control. Five days after implantation treatment was started using twice daily 20 mg/kg alisertib orally administered 5 days per week and/or 18 mg/kg TPI-287 administered intravenously every 4 days for a total of 3 treatments. Survival was assessed as well tumor volume using MR imaging at 2 weeks and 4 weeks after tumor implantation. Monotherapy with alisertib improved survival, which was further improved with the addition of TPI 287 (p=0.0058). TPI 287 alone did not significantly improve survival. Tumor volume was significantly decreased in all treatment groups at 2 weeks compared with control. At 4 weeks the alisertib and TPI 287 groups showed a trend toward decreased tumor volume with a significant decrease in the combination therapy group. This data supports the potential use of this combination therapy in human trials.

Unexplained neoplastic anastomotic recurrence after right hemicolectomy: a case report

Background Anastomotic recurrences of the colon are postulated to arise due to inadequate margins, tumor implantation by exfoliated cells, altered biological properties of bowel anastomosis, and missed synchronous lesions. In this paper, a case of unexpected early local recurrence after surgery for colon cancer is presented. Case presentation A 68-year-old Caucasian man underwent right hemicolectomy for invasive G2 adenocarcinoma. Two months later, endoscopy revealed a wide and well-functioning anastomosis with a hyperemic, hard, and thickened mucosal area of about 2 cm in diameter. Biopsies showed the presence of an adenocarcinoma with the same grading of the previous lesion. Ten days later, the patient underwent a new intervention; the last 10 cm of the ileum and half of the remaining transverse colon were resected, and the patient started adjuvant therapy. Specimen examination confirmed the presence of an adenocarcinoma (G2) penetrating the muscular layer of the wall; also, in this case, resection edges were free from tumoral invasion, and the removed lymph nodes were exempt from neoplastic colonization. The patient was seen in follow-up for about 5 years, and he did not show local or systemic manifestations. Conclusions Whenever a neoplastic recurrence on the anastomotic line occurs, in the presence of negative intestinal margins, as usual in right colectomies, the implantation of neoplastic cells could be the possible cause.