Injection Of Tumor Cells Research Articles

Abstract 569: Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME)

Abstract Targeting solid tumors with CAR T cells has proven to be more difficult due to their heterogenous target antigen expression, antigen escape, and due to their hostile tumor microenvironment (TME). Mesothelin represents a promising surface tumor antigen, since it has been associated with tumor invasion and is highly expressed on various cancer types, including pancreatic adenocarcinoma. As clinical trials of mesothelin targeting CAR have yet to show efficacy, we hypothesized that tumor stromal cells such as cancer-associated fibroblasts (CAFs) may play a role in this resistance. To provide a more favorable TME for CAR T cells, we generated a bicistronic lentiviral vector encoding a mesothelin CAR along with a secreted T cell engaging molecule (TEAM) that targets fibroblast activation protein (FAP), which is expressed by CAFs. We termed our constructs CARTEAM, and in this case, mesoFAP. We have assessed the activation and proliferative capacity of these CARTEAM through in vitro assays. We showed that TEAMs secreted by CAR T cells bind their appropriate target antigen by adding supernatant from CARTEAM to target cells expressing FAP. In a co-culture assay using a transwell system we demonstrated the cytotoxic effect of secreted TEAM interacting and recruiting bystander T cells against CAFs. In a real-time cell analysis (RTCA) co-culture assay with a pancreatic cancer cell line (AsPC1) and FAP expressing CAFs, we showed cell death of AsPC1 upon CAR recognition and cell death of CAFs through TEAM-mediated recruitment of bystander T cells and CART cells. We show in these co-culture systems, mimicking tumor and TME, that our CARTEAM construct is superior in the elimination of both cancer cell line and CAF, in comparison to control constructs, including mesothelin targeting CAR T cells (meso CAR) and meso CAR T cells secreting an unspecific CD19 TEAM (mesoCD19). We also used acoustic force microscopy to evaluate the additive effect of the TEAM molecule secreted to binding to tumor cells by mesothelin CAR T cells. In vivo experiments of subcutaneously injected tumor cells admixed with CAFs, show superior tumor control when treated with CARTEAM in comparison to control constructs. Based on these data, we demonstrate both the effective in vitro elimination of CAFs and pancreatic cancer cells through the application of CARTEAM and control of pancreatic tumor growth in vivo. Our studies provide a deeper insight into a dual targeting strategy using a novel CAR T cell secreting a TEAM against pancreatic cancer and its tumor microenvironment. Citation Format: Marc Wehrli, Adam Kuo, Rebecca Larson, Irene Scarfò, Amanda Bouffard, Korneel Grauwet, Mark Leick, Andrea Schmidts, Stefanie Bailey, Tamina Kienka, Michael Kann, Sonika Vatsa, Harrison Silva, Kathleen Gallagher, Max Jan, Bryan Choi, David Ting, Marcela Maus. Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 569.

Exploiting GLAAD molecules to drive an antitumor immune response in a colorectal cancer mouse model.

2565 Background: Only a minority of colorectal cancer (CRC) patients benefit from immune checkpoint inhibitors (ICI), with less than half of eligible patients responding to the treatment. Abnormal glycosylation of tumor cells is a signature of cancer development. Here, we described novel immunomodulatory molecules, named GLAAD (glycopeptides with adjuvant and tumor-associated antigen delivery). We hypothesized that GLAAD, by sharing similarities with aberrant glycans expressed in many cancers, may be a safe and effective treatment strategy to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a CRC mouse model, we tested whether two GLAAD candidates reduce tumor burden when orally administered alone or in combination with ICI. Methods: C57/BL6J mice were supplemented with 3% GNU-201 or 3% GNU-101 in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice received normal drinking water. On day 0, all mice were injected subcutaneously with MC38 CRC cells in each flank. On days 6, 9, 12, and 15, mice in each group received anti-PD1 antibody (aPD1) or an isotype control. Tumor size was measured every 3rd day. On day 18, the mice were sacrificed. Results: The aPD1 therapy alone showed a limited anti-tumor efficacy overall. Groups treated with GNU-201, GNU-101, GNU-201+aPD1 or GNU-101+aPD1 exhibited reduced growth, resulting in a significantly reduced tumor volume vs control on day 18. In contrast, GLAAD candidates alone significantly reduced tumor volume compared with control and led to a numerically larger decrease than aPD1 treatment alone, suggesting a higher efficacy. When analyzing tumor infiltrating myeloid cells on day 18, no differences were seen for neutrophils or dendritic cells but the proportion of macrophages was decreased in mice receiving GNU-201 or GNU-101. GNU-101 resulted in reduced serum levels of IL-10, a molecule participating in the suppression of antitumor immune responses. The tested GLAAD candidates stimulated robust anti-tumor T cell responses. GNU-201 and GNU-101 supplementation alone or in combination with aPD1 increased the proportion of T cells and among those CD8+ T cells, IFNγ producing CD4+ T cells, IFNγ+ and perforin+ CD8+ T cells. GNU-101 and GNU-201 elicited memory T cell responses after priming of the mice, measured as the ex vivo recall response (CD69 expression) of CD8+ T cells isolated from the tumor. Conclusions: GNU-201 and GNU-101 as standalone treatment induce a strong anti-tumor response that is as good as or even better than aPD1 alone. The therapeutic efficacy is associated with increased tumor infiltration and activation of T cells, in particular CD8+ T cells, highlighting the role of these GLAAD candidates as potent adjuvants in supporting specific tumor-associated antigen recognition by the immune system.

Exercise and Prebiotic Fiber Provide Gut Microbiota-Driven Benefit in a Survivor to Germ-Free Mouse Translational Model of Breast Cancer.

Simple SummaryBreast cancer is the most common cancer in women worldwide. In recent years, the community of microbes that inhabit the intestinal tract, called the gut microbiota, has been shown to influence patient response to several cancer therapies. On the other hand, treatments such as chemotherapy can disrupt the resident gut microbiota and potentially contribute to poor health outcomes. Strategies to improve the composition of the gut microbiota include dietary and exercise interventions. While diet and exercise are already established as important for breast cancer prevention, during treatment, and for reducing recurrence, little is known about the impact of these factors on the gut microbiota in the context of breast cancer. Therefore, our aim was to examine the impact of exercise and diet on the gut microbiota in breast cancer. Our findings indicate that exercise and prebiotic fiber supplementation may provide benefits to individuals with breast cancer through advantageous gut microbial changes. Our findings of a potential adjuvant of exercise and prebiotics should inspire further mechanistic and clinical investigations.The gut microbiota plays a role in shaping overall host health and response to several cancer treatments. Factors, such as diet, exercise, and chemotherapy, can alter the gut microbiota. In the present study, the Alberta Cancer Exercise (ACE) program was investigated as a strategy to favorably modify the gut microbiota of breast cancer survivors who had received chemotherapy. Subsequently, the ability of post-exercise gut microbiota, alone or with prebiotic fiber supplementation, to influence breast cancer outcomes was interrogated using fecal microbiota transplant (FMT) in germ-free mice. While cancer survivors experienced little gut microbial change following ACE, in the mice, tumor volume trended consistently lower over time in mice colonized with post-exercise compared to pre-exercise microbiota with significant differences on days 16 and 22. Beta diversity analysis revealed that EO771 breast tumor cell injection and Paclitaxel chemotherapy altered the gut microbial communities in mice. Enrichment of potentially protective microbes was found in post-exercise microbiota groups. Tumors of mice colonized with post-exercise microbiota exhibited more favorable cytokine profiles, including decreased vascular endothelial growth factor (VEGF) levels. Beneficial microbial and molecular outcomes were augmented with prebiotic supplementation. Exercise and prebiotic fiber demonstrated adjuvant action, potentially via an enhanced anti-tumor immune response modulated by advantageous gut microbial shifts.

Captopril, a Renin-Angiotensin System Inhibitor, Attenuates Tumour Progression in the Regenerating Liver Following Partial Hepatectomy.

(1) Liver regeneration following partial hepatectomy for colorectal liver metastasis (CRLM) has been linked to tumour recurrence. Inhibition of the renin–angiotensin system (RASi) attenuates CRLM growth in the non-regenerating liver. This study investigates whether RASi exerts an antitumour effect within the regenerating liver following partial hepatectomy for CRLM and examines RASi-induced changes in the tumour immune microenvironment; (2) CRLM in mice was induced via intrasplenic injection of mouse colorectal tumour cells, followed by splenectomy on Day 0. Mice were treated with RASi captopril (250 mg/kg/day), or saline (control) from Day 4 to Day 16 (endpoint) and underwent 70% partial hepatectomy on Day 7. Liver and tumour samples were characterised by flow cytometry and immunofluorescence; (3) captopril treatment reduced tumour burden in mice following partial hepatectomy (p < 0.01). Captopril treatment reduced populations of myeloid-derived suppressor cells (MDSCs) (CD11b+Ly6CHi p < 0.05, CD11b+Ly6CLo p < 0.01) and increased PD-1 expression on infiltrating hepatic tissue-resident memory (TRM)-like CD8+ (p < 0.001) and double-negative (CD4-CD8-; p < 0.001) T cells; (4) RASi reduced CRLM growth in the regenerating liver and altered immune cell composition by reducing populations of immunosuppressive MDSCs and boosting populations of PD-1+ hepatic TRMs. Thus, RASi should be explored as an adjunct therapy for patients undergoing partial hepatectomy for CRLM.

Endometrial Cancer Patient-Derived Xenograft Models: A Systematic Review.

Background: Because patient-derived xenograft (PDX) models resemble the original tumors, they can be used as platforms to find target agents for precision medicine and to study characteristics of tumor biology such as clonal evolution and microenvironment interactions. The aim of this review was to identify articles on endometrial cancer PDXs (EC-PDXs) and verify the methodology and outcomes. Methods: We used PubMed to research and identify articles on EC-PDX. The data were analyzed descriptively. Results: Post literature review, eight studies were selected for the systematic review. Eighty-five EC-PDXs were established from 173 patients with EC, with a total success rate of 49.1%. A 1–10 mm3 fragment was usually implanted. Fresh-fragment implantation had higher success rates than using overnight-stored or frozen fragments. Primary tumors were successfully established with subcutaneous implantation, but metastasis rarely occurred; orthotopic implantation via minced tumor cell injection was better for metastatic models. The success rate did not correspond to immunodeficiency grades, and PDXs using nude mice reduced costs. The tumor growth period ranged from 2 weeks to 13 months. Similar characteristics were observed between primary tumors and PDXs, including pathological findings, gene mutations, and gene expression. Conclusion: EC-PDXs are promising tools for translational research because they closely resemble the features of tumors in patients and retain molecular and histological features of the disease.

T cell receptor revision as a novel mechanism of action of immune checkpoint blockade

Abstract Background Immune checkpoint blockade (ICB) is a promising therapy prescribed to combat tumors. However, acquired resistance to ICB highlights the need for a better understanding of ICB mechanisms. Analysis of T cell receptor (TCR) sequences in individuals before and after ICB showed that a distinct TCR repertoire was observed following ICB, suggesting that T cells with novel specificities contribute to tumor clearance. While the source of these T cells with distinct TCRs is not clear, ICB has been found to lead T cells infiltrating both mouse and human tumors to express components of TCR revision. Therefore, one potential mechanism by which ICB leads to anti-tumor immunity is by inducing TCR revision. Methods Wildtype C57BL/6, as well as TCR revision-deficient TCRa+/−.Rag2fl/fl.CD4cre (TCRa+/−.Rag2CD4) mice received subcutaneous injections of either 4.5 × 105 B16F10 or MC38F10 cells. Mice then received either PBS or a combination of 250 μg each of anti-CTLA-4, anti-PD-1, and anti-PDL-1 three times per week starting Day 7 after tumor cell injection. Tumor size and mouse survival were monitored, and baseline splenic and thymic immune cell numbers were measured via flow cytometry. Results No differences in T or B cell subsets were detected at baseline between the mouse strains. ICB significantly reduced tumor growth and mortality in the wildtype, but not the TCRa+/−.Rag2CD4, mice for both tumor models tested. Conclusions TCR revision capabilities seem to be important in ICB efficacy. Further investigation using these mouse models will help explore the possibility of future targeting of TCR revision pathways to increase ICB efficacy.

Zebrafish Patient-Derived Avatars from Digestive Cancers for Anti-cancer Therapy Screening.

Patient-derived xenografts (PDXs), also called "avatars," are generated by the implantation of human primary tumor cells or tissues into a host animal. Given the complexity and unique characteristics of each tumor, PDXs are models of choice in cancer research and precision medicine. In this context, the zebrafish PDX model (zPDX or zAvatar) has been recognized as a promising in vivo model to directly challenge patient cells with anti-cancer therapies in a personalized manner. The assay relies on the injection of tumor cells from patients into zebrafish embryos to then test and identify the best available drug combination for a particular patient. Compared to mouse PDXs, zAvatar assays take less time and do not require in vitro or in vivo cell expansion. The present article describes how to generate zAvatars from resected digestive cancer from surgeries and how to then use them for anti-cancer therapy screening. We describe the steps for tumor sample collection and cryopreservation, sample preparation and fluorescent labeling for microinjection into zebrafish embryos, drug administration, and analysis of tumor behavior by single-cell confocal imaging. We provide detailed protocols and helpful tips for performing this assay, and we address the technical challenges associated with the workflow. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Patient tumor sample collection and cryopreservation Basic Protocol 2: Generation of zAvatars and anti-cancer treatment Basic Protocol 3: Whole-mount immunofluorescence Basic Protocol 4: Confocal imaging and analysis.

Immunomodulatory effect of splenectomy in lung cancer mouse xenograft models receiving radiation therapy.

PurposeThis study aims to investigate the effect of splenectomy on radiation-mediated growth inhibition and immune modulation in lung cancer xenograft models.Materials and MethodsHuman non-small cell lung cancer H1299 cells and murine Lewis lung carcinoma LL/2-luc cells were injected into the right hind leg of BALB/c-nude mice and C57BL/6 mice, respectively. Splenectomy or sham operation was performed prior to tumor cell injection or before and after irradiation during tumor growth. Irradiation was delivered with 2–3 fractions of 6 Gy X-ray using a linear accelerator. Flow cytometry analysis was performed for immune cell profiling.ResultsSplenectomy prior to tumor injection or at early stage inhibited growth of LL/2-luc tumors but not that of H1299 tumors; however, it did not enhance the antitumor effect of radiation regardless of intervention timing. Flow cytometry analysis showed monocytic myeloid-derived suppressor cells (MDSCs) and activated CD8+ T cells increased after irradiation in the tumors of splenectomized mice, compared to those of sham-operated mice. Administration of anti-PD-1 (programmed death-1) antibodies improved the ability of splenectomy to attenuate the growth of irradiated tumors.ConclusionSplenectomy has paradoxical effects on radiation-induced tumor growth inhibition, depending on tumor types and intervention timing, but it has an immune-modulating effect when combined with radiation.

Abstract P5-08-03: Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer

Abstract Background: Increased detection of ductal carcinoma in situ (DCIS) has been a consequence of mammographic screening. Currently there is a reappraisal of the necessity of aggressive treatment, with their attendant toxicities for a preneoplastic lesion with 15-20% chance of recurrence or progression. The recombinant immunotoxin, TFR(fv)PE40, binds to the abundantly expressed transferrin receptors on cancer cells, is internalized, processed and trafficked to the cytosol. The released Pseudomonas exotoxin (PE) catalyzes the transfer of ADP-ribose from NAD to elongation factor 2 (EF2), inactivates EF2, and leads to an arrest in protein translation, and cell death by apoptosis. However, TFR(fv)PE40 is toxic to liver cells. We hypothesized that, when administered via the opening at the mammary teat, the immunotoxin will destroy preneoplasias in the entire ductal tree. Because of the closed nature of the ductal system, the toxic effects of the TFR(fv)PE40 will be confined to the tumor cells alone, and systemic effects will be minimal or absent. The intraductal approach, we proposed, will provide a direct, safe and effective treatment for DCIS. Methods: We tested this concept in two models of DCIS using two luc-tagged cell lines-SUM225-luc, derived from a human DCIS, and in MCF-7-luc. In both cell lines, the IC50 of the toxin was in the nM range. DCIS develop within one week in MIND models following intraductal (i.duc) injection of tumor cells (right and left 4th mammary gland) of ex-breeder NSG mice. DCIS progressed to invasive cancer in 2-3 weeks. At day 7, mice were administered vehicle, TFR mAb alone, or the HB21(fv)PE40 conjugate at two doses (0.15 or 1.5 ug per teat x 2) by the i.duc route, 3 times at weekly intervals, and observed for 1-3 months. Mice treated with the TFR-Ab showed no significant reduction in tumor size. Treated with HB21(fv)PE40 i.duc, by IVIS imaging, tumors were undetectable in both models (P&amp;lt;0.0001) within two weeks. No recurrence was observed during 3 months of followup. Mammary whole mount analysis and histopathology showed that i.duc toxin rendered more than 90% of the mice cancer-free. Immunohistochemical analysis of tumor sections for huTFR (CD71), Ki67, and CD31 supported the loss of TFR-expressing and proliferating cancer cells, and reduction of blood vessels. Pharmacokinetics studies showed that toxin was detectable by TFR-ELISA in blood 5, 30 and 60 min after i.p injection, but was undetectable in the blood of i.duc-treated mice. Conclusions: Overall, we have shown that i.duc HB21(fv)PE40 has remarkable antitumor activity in cell culture with an IC50 in the nM range, and in two MIND models of DCIS. Blood levels of the toxin after i.duc injection were undetectable, suggesting that the toxin remains inside the ductal system during its 30 min half-life, and its effect occurs during this time. This preclinical study provides a strong basis for conducting human trials. The toxicity of HB21(Fv)PE40 by the i. duc route of drug administration could be determined in dose escalation studies in patients with DCIS and early breast cancer who will undergo mastectomy as part of their treatment regimen, using a previously successful study design (PMID:2230751). This design will allow detailed examination of the local and systemic effects of HB21(fv)PE40 administered i.duc and reintroduce a very potent agent into DCIS treatment, and in the future, cancer prevention in women at high risk of developing breast cancer. Citation Format: Saraswati Sukumar, Guannan Wang, Alok Kumar, Wanjun Ding, Preethi Korangath, Priya Pai, Kathleen Gabrielson, Ira Pastan. Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-03.

Development of a novel castration-resistant orthotopic prostate cancer model in New Zealand White rabbit.

Prostate cancer (PCa) models in mice and rats are limited by their size and lack of a clearly delineated or easily accessible prostate gland. The canine PCa model is currently the only large animal model which can be used to test new preclinical interventions but is costly and availability is sparse. As an alternative, we developed an orthotopic human prostate tumor model in an immunosuppressed New Zealand White rabbit. Rabbits are phylogenetically closer to humans, their prostate gland is anatomically similar, and its size allows for clinically-relevant testing of interventions. Rabbits were immunosuppressed via injection of cyclosporine. Human PC3pipGFP PCa cells were injected into the prostate via either (a) laparotomy or (b) transabdominal ultrasound (US) guided injection. Tumor growth was monitored using US and magnetic resonance imaging (MRI). Contrast-enhanced ultrasound (CEUS) imaging using nanobubbles and Lumason microbubbles was also performed to examine imaging features and determine the optimal contrast dose required for enhanced visualization of the tumor. Ex vivo fluorescence imaging, histopathology, and immunohistochemistry analyses of the collected tissues were performed to validate tumor morphology and prostate-specific membrane antigen (PSMA) expression. Immunosuppression and tumor growth were, in general, well-tolerated by the rabbits. Fourteen out of 20 rabbits, with an average age of 8 months, successfully grew detectable tumors from Day 14 onwards after cell injection. The tumor growth rate was 39 ± 25 mm2 per week. CEUS and MRI of tumors appear hypoechoic and T2 hypointense, respectively, relative to normal prostate tissue. Minimally invasive US-guided tumor cell injection proved to be a better method compared to laparotomy due to the shorter recovery time required for the rabbits following injection. Among the rabbits that grew tumors, seven had tumors both inside and outside the prostate, three had tumors only inside the prostate, and four had tumors exclusively outside of the prostate. All tumors expressed the PSMA receptor. We have established, for the first time, an orthotopic PCa rabbit model via percutaneous US-guided tumor cell inoculation. This animal model is an attractive, clinically relevant intermediate step to assess preclinical diagnostic and therapeutic compounds.

Burrowing behaviour of rats: Strain differences and applicability as well-being parameter after intracranial surgery.

In mice, burrowing is considered a species-typical parameter for assessing well-being, while this is less clear in rats. This exploratory study evaluated burrowing behaviour in three rat strains during training and in the direct postoperative phase after complex intracranial surgery in different neuroscience rat models established at Hannover Medical School or Aachen University Hospital. Male Crl:CD (SD; n = 18), BDIX/UlmHanZtm (BDIX; n = 8) and RjHan:WI (Wistar; n = 35) rats were individually trained to burrow gravel out of a tube on four consecutive days. Thereafter, BDIX rats were subjected to intracranial injection of BT4Ca cells and tumour resection (rat glioma model), SD rats to injection of 6-hydroxydopamine (6-OHDA) or vehicle (rat Parkinson's disease model) and Wistar rats to endovascular perforation or sham surgery (rat subarachnoid haemorrhage (SAH) model). Burrowing was retested on the day after surgery. During training, BDIX rats burrowed large amounts (mean of 2370 g on the fourth day), while SD and Wistar rats burrowed less gravel (means of 846 and 520 g, respectively). Burrowing increased significantly during training only in Wistar rats. Complex surgery, that is, tumour resection (BDIX), 6-OHDA injection (SD) and endovascular perforation or sham surgery for SAH (Wistar) significantly reduced burrowing and body weight, while simple stereotactic injection of tumour cells or vehicle did not affect burrowing. Despite the training, burrowing differed between the strains. In the direct postoperative phase, burrowing was reduced after complex surgery, indicating reduced well-being. Reduced burrowing was accompanied with postoperative weight loss, a validated and recognised quantitative measure for severity assessment.

Inhibition of integrin αvβ6 sparks T-cell antitumor response and enhances immune checkpoint blockade therapy in colorectal cancer

BackgroundIntegrin αvβ6 is a heterodimeric cell surface protein whose cellular expression is determined by the availability of the integrin β6 subunit (ITGB6). It is expressed at very low levels in...

Dexmedetomidine Associated Lung Cancer Proliferation and Tumor Growth is Prevented by β-Caryophyllene: Role of AMPK Activation and PGC-1α/TFAM Over Expression

Background: Dexmedetomidine has been reported to induce anti-apoptotic effects and metastatic progression in lung cancer. In the current investigation, the effect of β-Caryophyllene on dexmedetomidine induced cell proliferation and apoptosis of lung cancer cells and tumor growth in mice was studied. Methods: A549 cell line was cultured with either dexmedetomidine alone or together with β-Caryophyllene for 24 h and analysed for cell proliferation with MTT assay. ELISA based kit was used to determine apoptotic DNA fragmentation. Western blotting was used to determine expression levels of target proteins. The induction of experimental lung tumor in rat model was achieved through the injection of A549 tumor cells subcutaneously into the middle left side of the mice after anesthetization with pentobarbital (35 mg/kg) at 2.8 × 106 cells in 400 μl of PBS. Result: We found that β-Caryophyllene exerts the anti-proliferative effects on A549 cells. Furthermore, β-Caryophyllene significantly prevents apoptotic cell death and causes up-regulation of PGC-1α and TFAM compared to dexmedetomidine treated cells. We observed that β-Caryophyllene suppressed tumor development in mice significantly compared to dexmedetomidine treated group without changing body weight.

Фенотипическое разнообразие макрофагов при раке яичников

Фенотипическое разнообразие макрофагов при раке яичников

Protocols for the Evaluation of a Lymphatic Drug Delivery System Combined with Bioluminescence to Treat Metastatic Lymph Nodes.

Bioluminescence (BL) imaging is a powerful non-invasive imaging modality widely used in a broad range of biological disciplines for many types of measurements. The applications of BL imaging in biomedicine are diverse, including tracking bacterial progression, research on gene expression patterns, monitoring tumor cell growth/regression or treatment responses, determining the location and proliferation of stem cells, and so on. It is particularly valuable when studying tissues at depths of 1 to 2cm in mouse models during preclinical research. Here we describe the protocols for the therapeutic evaluation of a lymphatic drug delivery system (LDDS) using an in vivo BL imaging system (IVIS) for the treatment of metastatic lymph nodes (LNs) with 5-fluorouracil (5-FU). The LDDS is a method that directly injects anticancer drugs into sentinel LNs (SLNs) and delivers them to their downstream LNs. In the protocol, we show that metastases in the proper axillary LN (PALN) are induced by the injection of luciferase-expressing tumor cells into the subiliac LN (SiLN) of MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice. 5-FU is injected using the LDDS into the accessory axillary LN (AALN) to treat tumor cells in the PALN after the tumor cell growth is confirmed in the PALN. The tumor growth and therapeutic effects are evaluated by IVIS. This method can be used to evaluate tumor growth and efficacy of anticancer drugs/particles, radiotherapy, surgery, and/or a combination of these methods in various experimental procedures in the oncology field.

"Smart" nanoscalpel for microsurgery of glial tumors of the human brain

We studied the effectiveness of magnetomechanical therapy in the treatment of brain glial tumors using magnetic nanodiscs functionalized with DNA aptamers to human brain tumor glial cells. • Materials and methods. The formation of a model of human glioblastoma was carried out by intracranial injection of tumor cells of glioblastoma obtained from a patient with glioblastoma. Antitumor therapy was carried out using nanodiscs modified with the Gli233 aptamer. The growth of the glial tumor was monitored using NMR tomography. • Results and discussion. Therapy of a glial tumor during 4 sessions of magnetomechanical therapy using a "smart" nanoscalpel in MF (10Hz, 100Oe) led to a significant reduction in its size, while glial tumors in mice that were treated with nanodiscs modified with nonspecific aptamers continued to increase in size. • Conclusion. Microsurgery using three-layer magnetic nanodisks with a quasi-dipole structure (Au/Ni/Au) modified with the specific for glial cells Gli233 aptamer (“smart” nanoscalpel) is effective for the treatment of human glial tumors in the brain.

PYCR1: A Potential Prognostic Biomarker in Pancreatic Ductal Adenocarcinoma

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor with an extremely poor prognosis in digestive tumors. Pyrroline-5-carboxylate reductase 1 (PYCR1) plays an important role in tumor development. Therefore, we aimed to explore the effect of PYCR1 on the growth of PDAC cells.Methods: Tumor tissues and adjacent normal pancreatic tissues were collected from 89 patients with PDAC. And immunohistochemistry (IHC) was used to analyze the expression level of PYCR1 in both. RNA interference was used to inhibit the expression of PYCR1 in PANC- 1 and AsPC-1 cells. After infection, the expression of PYCR1 protein was detected by Western blot. The proliferation and growth of PDAC cells were detected by Celigo analysis, MTT, and clone formation assay. Cell apoptosis was analyzed by flow cytometry. Furthermore, the effect of PYCR1 interference on tumor growth was evaluated in vivo through injecting tumor cells subcutaneously into nude mice.Results: The expression of PYCR1 in pancreatic cancer tissues was significantly higher than in paired adjacent normal pancreatic tissues (P <0.01). In vitro, the downregulation of PYCR1 expression significantly inhibited the cell proliferation and colony formation, and increased apoptosis in PANC-1 cells and AsPC-1 cells compared with the shCtrl group (P <0.01). And in vivo, PYCR1 interference also significantly inhibited tumor growth both in the tumor volume and weight.Conclusion: PYCR1 interference was able to inhibit cell proliferation and promote cell apoptosis of pancreatic cancer. The PYCR1 may serve as a potential therapeutic and prognostic biomarker for the treatment of pancreatic cancer.

Establishment of In Vivo Ovarian Cancer Mouse Models Using Intraperitoneal Tumor Cell Injection.

Mouse models-xenograft models, syngeneic models (directly implanted or chemically or virally induced), and genetically engineered mice (including transgenic and knockout methods) are invaluable for preclinical studies of ovarian cancer as they recapitulate the structures and microenvironments of tumors, which in vitro studies are unable to accomplish.This chapter describes the methodology and approaches for generating various murine models currently employed in ovarian cancer research. It covers the implantation of cells from ovarian cancer cell lines into mice by intraperitoneal injection.

Tasquinimod Targets Immunosuppressive Myeloid Cells, Increases Osteogenesis and Has Direct Anti-Myeloma Effects By Inhibiting c-Myc Expression in Vitro and In Vivo

IntroductionImmunotherapy has revolutionized cancer treatment and significantly affected the management of Multiple Myeloma (MM) patients. Unfortunately, these immunotherapeutic approaches are hampered by the presence of a suppressive bone marrow microenvironment including myeloid derived suppressor cells and tumor associated macrophages. Tasquinimod (TasQ), an immunomodulatory compound, is currently in phase Ib/IIa for relapsed/refractory MM patients (NCT04405167). TasQ blocks the interaction between S100A9 and its receptors, which is associated with reduced MDSC accumulation. In this study, we investigated TasQ-mediated direct and indirect effects on MM cell growth, bone disease and immunomodulation in vitro and in vivo using human myeloma cell lines and the immunocompetent 5TMM models.Material and methodsIn vitro, murine (5T33vt, 5TGM1) and human (JJN3, LP1, OPM2, and RPMI8226) MM cell lines were cultured at different concentrations of TasQ. Cell proliferation was assessed by BrdU staining using flow cytometry. C-Myc and pSTAT3 expression were analyzed by western blot. In vitro T cell proliferation experiments were performed using MACS-sorted CD11b + cells and CFSE-labeled T cells from naïve mice. Cells were cocultured for 72h in the presence of MM conditioned medium (5T33MMvt CM) with CD3/CD28 microbeads, followed by flow cytometry to assess T cell proliferation. For in vivo experiments, we used the 5T33 (aggressive) and 5TGM1 (moderate) MM models. On the second day after tumor cell injection, the mice were randomly assigned to the treatment group and the control group. The treatment group received 30 mg/kg of TasQ in drinking water for 35 days (5TGM1) and 21 days (5T33). Anti-tumor and immunomodulating effects were analyzed by flow cytometry (e.g. tumor cells, myeloid subsets, CD4/CD8 + T cells), qRT-PCR, western blot and serum ELISA (interferon-gamma). Effects on osteogenesis in the 5TGM1 model was investigated by Micro-CT. Statistical differences were assessed by Mann-Whitney U test and One-way ANOVA with p<0.05 considered as statistically significant.ResultsTasQ-treatment of murine and human myeloma cell lines (HMCL), at concentrations of 10-25uM, significantly reduced MM cell proliferation after 24h and 48h in vitro (n=3, p<0.05). In addition, a downregulation in c-Myc expression could be observed 6h after treatment of human MM cell lines (n=3). In vitro, TasQ significantly increased T cell proliferation in co-culture experiments with T cells and myeloid cells in 5T33MMvt CM (n=3, p<0.05).Using the immunocompetent 5TGM1 and 5T33MM model, we investigated direct and indirect anti-tumor effects of TasQ. We found that TasQ significantly reduced tumor load in the bone marrow of 5TGM1 (n=10/group, p=0.0012) and 5T33MM mice (n=10/group, p=0.0106) compared to vehicle-treated control mice. Using flow cytometry, we could not observe a difference in the percentage of CD4 + and CD8 + T cells. However, a significant upregulation in serum interferon-gamma could be observed in the 5T33MM mice (p=0.0284). While the percentage of CD11b + cells in the TasQ-treated group was significantly increased (p<0.05), the percentage of monocytic myeloid cells (CD11b +Ly6G -) was significantly reduced in both models (p<0.05). qRT-PCR results showed that the expression of IL-10 was downregulated in purified CD11b + myeloid cells (p<0.05). Consistent with the in vitro data, we observed a decrease in the protein expression of c-Myc in purified MM cells obtained from TasQ-treated mice compared to control mice. Micro-CT analysis of femurs demonstrated a significant increase in the percentage BV/TV (ratio of bone material volume over tissue volume) and trabeculae number (p<0.0001) in TasQ-treated 5TGM1 mice compared to untreated mice.ConclusionTasQ has pleiotropic effects on the MM cells and its surrounding bone marrow microenvironment. It affects MM cell growth by decreasing c-Myc expression. In addition, TasQ targets the immunosuppressive monocytic myeloid cell population and increases serum interferon-gamma levels, indicative for immune cell activation. Moreover, it stimulates osteogenesis in vivo. Taken together, all these data provide evidence for the therapeutic benefits of TasQ as an anti-MM therapy for patients. DisclosuresTörngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. De Veirman: Active Biotech AB: Research Funding.

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.