It is provided the use of oncolytic VSVd51-hGM-CSF for the treatment of a solid cancer.
Bladder cancer (BC) is consistently among the six most prevalent cancers in men with approximately 500,000 new cases diagnosed annually worldwide. BC poses significant clinical problems and is the most expensive solid cancer to treat due to the requirement for specialized monitoring equipment and highly trained personnel. Most BC cases are non-muscle invasive (NMIBC), but they are associated with high recurrence rates and a significant risk of progression to muscle invasive disease. Transurethral resection (TUR) along with intravesical chemo- or immunotherapies have been shown to decrease the recurrence and/or progression rates of NMIBC. However, despite these front-line therapies, 50-80% of patients will develop recurrence at 5 years, of which up to 25% will evolve into muscle invasive BC (MIBC). For high-risk NMIBC, the use of intravesical Bacillus Calmette-Guérin (BCG) has shown significant benefits in reducing recurrence and progression compared to TUR alone. Unfortunately, up to 84% of patients are unable to complete the 3 year BCG regimen due to treatment inefficacy and local and/or systemic toxic effects. Cystectomy remains the standard of treatment for high-risk patients who have failed BCG therapy. Patients who undergo cystectomy before their bladder cancer progresses to muscle invasive disease, have shown good disease free survival. However, cystectomy poses a significantly diminished quality of life.
Recent studies have shown that BCG and interferon (IFNα) combination therapy may be useful as salvage regimen in BCG failures. However, various tumors acquire defects in their ability to respond to IFNs as they evolve and many aggressive BC cell lines are highly resistant to IFN treatment. IFN-resistance confers a growth advantage for cancer cells over normal tissues, but simultaneously compromises their antiviral response. To exploit this vulnerability of aggressive BC cells, Zhang et al. (2010, Int J Cancer, 127: 830-838) used an oncolytic rhabdovirus—Vesicular Stomatitis Virus (VSV) that possesses the ability to selectively infect, replicate in and kill IFN-resistant BC cells, but is strongly suppressed in IFN-responsive normal tissues. The tumor specificity of wild-type VSV has been further enhanced in an attenuated strain (VSVd51) with a point mutation in the matrix protein, which has a defect in its ability to short-circuit the antiviral activity of IFNs, and induces an enhanced protective response in normal tissues, while maintaining its oncolytic ability. Importantly, the lack of pre-existing neutralizing antibodies in human populations, a major hurdle that impedes the in vivo delivery of many other oncolytic viruses (OVs, e.g. adeno-, herpes, vaccinia, measles virus), warrants the development of oncolytic rhabdoviruses for clinical applications.
It is thus highly desirable to be provided with alternative bladder-sparing therapies that are effective and less toxic for high-risk BC to prevent both recurrence and progression.
It is provided a composition comprising (i) VSVd51-hGM-CSF construct as depicted in SEQ ID NO: 1, (ii) a functional equivalent thereof, or (iii) a nucleotide sequence having at least 70% identity with SEQ ID NO: 1; and a carrier.
In an embodiment, the composition described herein is for treating a solid cancer.
In another embodiment, the solid cancer is bladder cancer, pancreatic cancer, breast cancer, colorectal cancer, ovarian cancer, or melanoma.
In a particular embodiment, the solid cancer is bladder cancer.
In a further embodiment, the composition is formulated for an administration selected from parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal and intramuscular.
It is also provided a method of treating a solid cancer comprising administering to a patient in need thereof a composition comprising a vesicular stomatitis virus (VSV) and a growth factor, wherein preferably, the VSV is VSVd51, the growth factor is a human growth factor, and the human growth factor is granulocyte macrophage-colony stimulating factor (GM-CSF).
In an embodiment, the composition comprises VSVd51-hGM-CSF construct as depicted in SEQ ID NO: 1.
It is provided that the composition is used as a monotherapy or in combination with an immunomodulator and/or Bacillus Calmette-Guérin (BCG).
In an embodiment, the immunomodulator is a type I interferon.
In an embodiment, the immunomodulator is an interferon alpha (IFNα).
In another embodiment, the composition increases expression of at least one of CD80, CD86, HLA-DR and PD-L1 in said patient.
In a further embodiment, the composition increases ATP levels in said patient.
In a supplemental embodiment, the composition increases gene expression of at least one of CCL4, CCL5, CXCL9, CXCL10, CXCL11, IFNγ, IL6, IRF-1, CSF-2, TNFα, CSF-2, TAP1 and TAP2 compared to untreated controls.
It is also provided the use of a composition as described herein for treating a solid cancer in a patient in need thereof.
Reference will now be made to the accompanying drawings.
In accordance with the present description, there is provided the use of oncolytic VSVd51-hGM-CSF for the treatment of bladder cancer.
It is provided administering a virus (vesicular stomatitis virus, VSV) that is not a common human pathogen to reduce the viability of bladder cancer cells, while leaving normal cells largely unharmed. The encompassed virus has been engineered to contain a special human growth factor (granulocyte macrophage-colony stimulating factor, GM-CSF) that will stimulate an immune response by attracting and promoting the development of antigen presenting cells and effector immune cells. The immune response will help with the local removal of bladder cancer cells as well cancer cells that may have spread to regional lymph nodes or other organs (metastases).
Given the urgent medical need for the development of additional and less toxic bladder-sparing therapies for BC patients failing frontline treatments, a novel VSVd51 containing human GM-CSF (VSVd51-hGM-CSF) is provided. Using both the human and existing mouse variant (VSVd51-mGM-CSF), their ability to treat BC was evaluated. BC cell lines were assessed for susceptibility to viral lysis and expression of ICD markers and immune gene signatures.
Due to the therapeutic potential of GM-CSF, a human GM-CSF transgene was incorporated into the backbone of the oncolytic VSVd51 variant to create VSVd51-hGM-CSF (
There is strong evidence to suggest that the immune system plays a critical role in determining the outcome of VSV therapy. Given the importance of the mode of tumor cell death in initiating anti-tumor immune response, ICD was assessed following infection of the mouse MB49 cell line with VSVd51 or VSVd51-mGM-CSF. High mobility group box 1 (HMGB1) protein was first measured (
A panel of genes related to pro-inflammatory, anti-inflammatory, antigen presentation and immune differentiation markers was examined by qPCR. Twenty-four hours following infection with the viruses, a general upregulation of genes related to immune cell recruitment and activation was detected in MB49 cells. Notably, mouse CCL2, CCL5, CXCL2, CXCL10 and GM-CSF transcripts showed an increase in expression in MB49 cells following VSVd51-mGM-CSF infection compared to VSVd51 and non-infected controls (
To determine if the observed in vitro ICD and immune gene signatures in mouse BC cells, translate to improved immune function in vivo, VSVd51 and VSVd51-mGM-CSF were compared in the treatment of C57BI/6 mice bearing orthotopic MB49 tumors (
In contrast, both the proportion and function of bladder infiltrating NK and CD8+ T cells were significantly increased in VSVd51-mGM-CSF treated mice (
To further investigate whether the improved immune function of treated mice will result in better disease outcome, mice were monitored for survival and measured tumor volume by small animal ultrasound. Reduced tumor volume and improved survival was observed in VSVd51-mGM-CSF-treated mice compared to controls (
To test the translational potential of VSVd51-hGM-CSF, its anti-cancer effect was examined on human BC cell lines and primary human immune cells. To do so, the human 5637 and UM-UC-3 BC cell lines was propagated as 3D spheroids instead of 2D monolayers to better mimic the physiology of the bladder urothelium. Using these spheroids, biomarkers of ICD were examined including secreted HMGB1 and ATP following infection. In the cell-free supernatants of VSVd51-hGM-CSF infected 5637 spheroids, increased levels of HMGB1 were observed, while similar HMGB1 levels were observed in UM-UC-3 spheroids (
In co-culture experiments with CD14+ human monocytes incubated with cell-free lysates from infected 5637 spheroids, polarization of these monocytes was observed towards an M1-like phenotype expressing higher levels of CD80, CD86, HLA-DR and PD-L1 that have been previously suggested to promote anti-tumor immune responses (
BC patients were enrolled in the observational oVSV-bladder study (Ethics protocol #2018-2414). To better model the physiological structures of the bladder urothelium, these BC patient were propagated derived tissue as 3D organoids ex vivo. Organoids from patient 34 and 38 were infected with VSVd51-hGM-CSF for 6 h and qPCR for gene expression analysis and assays to measure biomarkers of ICD were conducted. Patient 34 displayed an immunogenic gene expression pattern with enhanced expression of multiple immune genes, notably CCL4, CCL5, CXCL9, CXCL10, CXCL11, IFNγ, IL6, IRF-1 and CSF-2 compared to untreated controls. In patient 38—CXCL9, CXCL10, CXCL11, IFNγ, IRF-1, TNFα and CSF-2 gene expression were also increased (
Moving beyond immune gene signatures, biomarkers of ICD were compared in VSVd51 infected vs. VSVd51-hGM-CSF infected BC organoids. Biomarkers of ICD including HMGB1 release for both patients was detected at higher levels in VSVd51-hGM-CSF infected organoids compared to VSVd51 infected and uninfected controls (
Accordingly, from in vitro experiments, it was determined that infection of mouse and human BC cells with VSVd51-mGM-CSF and VSVd51-hGM-CSF, respectively, results in higher ICD compared to VSVd51 infected and non-infected cells. Enhanced release of intracellular HMGB1, ATP and increased Calreticulin+/DAPI+ tumor cell populations (
Interestingly, while the overall survival in VSVd51-mGM-CSF treated mice was improved, there was a proportion of mice who developed large bladder tumors and succumbed to disease faster than the VSVd51 treated group. Upon closer examination, accumulation of ARG1+ gMDSC was observed in the bladder tumor tissue of VSVd51-mGM-CSF treated mice (
In translational studies, an analogous mechanism of VSVd51-hGM-CSF-induced immune activation is occurring in human BC spheroids and patient organoids. VSVd51-hGM-CSF infection of human BC spheroids resulted in the enhanced release of immunogenic DAMPs, polarization of human monocytes towards an M1-like phenotype and lead to greater NK and CD8+ T cell migration (
It is therefore provided a composition comprising a VSVd51-hGM-CSF construct as depicted in SEQ ID NO: 1 and a carrier.
The composition provided herewith can be administered by any means, such as e.g. by a route of administration selected from parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal and intramuscular.
In an embodiment, the composition described herein could be administered with an immunomodulator (e.g. type I interferon, more preferably interferon alpha (IFNα)) and/or Bacillus Calmette-Guérin (BCG) to said patient. It is encompassed that the composition described herein can be used to treat solid cancer, such as e.g. bladder cancer, pancreatic cancer, breast cancer, colorectal cancer, ovarian cancer, and melanoma.
The terms “functional equivalents” and “functional variants” are used interchangeably herein and such functional equivalents of VSVd51-hGM-CSF are encompassed herein. Functional nucleic acid equivalents may typically contain silent mutations or mutations that do not alter the biological function.
As defined herein, the term “substantially homologous” refers to a first nucleotide sequence which contains a sufficient or minimum number of identical or equivalent nucleotides to a second nucleotide sequence such that the first and the second nucleotide sequences have a common domain. For example, nucleotide sequences which contain a common domain having about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or more are defined herein as sufficiently identical.
As used herein, “pharmaceutical composition” means therapeutically effective amounts (dose) of an agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. A “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions may be liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, and detergents (e.g., Tween™ 20, Tween™ 80, Pluronic® F68, bile acid salts). Compositions of the invention may also comprise solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), and bulking substances or tonicity modifiers (e.g., lactose, mannitol) which contribute to covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the active agent. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention include particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral routes.
In addition, the term “pharmaceutically effective amount” or “therapeutically effective amount” refers to an amount (dose) effective for treating a patient, having, for example, a nerve injury. It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route or taken alone or in combination with other therapeutic agents.
A therapeutically effective amount or dosage of an active agent may range from about 0.001 to 30 mg/kg body weight, with other ranges of the invention including about 0.01 to 25 mg/kg body weight, about 0.025 to 10 mg/kg body weight, about 0.3 to 20 mg/kg body weight, about 0.1 to 20 mg/kg body weight, about 1 to 10 mg/kg body weight, 2 to 9 mg/kg body weight, 3 to 8 mg/kg body weight, 4 to 7 mg/kg body weight, 5 to 6 mg/kg body weight, and 20 to 50 mg/kg body weight. In other embodiments, a therapeutically effective amount or dosage of an active agent may range from about 0.001 to 50 mg total, with other ranges of the invention including about 0.01 to 10 mg, about 0.3 to 3 mg, about 3 to 10 mg, about 6 mg, about 9 mg, about 10 to 20 mg, about 20-30 mg, about 30 to 40 mg, and about 40 to 50 mg.
The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an active compound can include a single treatment or a series of treatments. In one example, a subject is treated with an active compound in the range of between about 0.3 to 10 mg, one time per week for between about 1 to 10 weeks, alternatively between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of an active compound used for treatment may increase or decrease over the course of a particular treatment.
In an embodiment, the composition described herein is for treating a solid cancer.
In addition to the provided results herein, as seen in Table 1, the composition described herein was used in known cancer cell lines:
In another embodiment, the solid cancer is bladder cancer, pancreatic cancer, breast cancer, colorectal cancer, ovarian cancer, or melanoma.
MB49 were maintained in DMEM; 5637 in RPMI; UM-UC-3 in EMEM, all supplemented with 10% HI FBS+100 U/ml penicillin and 100 μg/ml streptomycin (complete media). 5637 and UM-UC-3 cell lines were purchased from ATCC and MB49 cell line from Millipore-Sigma and were verified to be mycoplasma free and show appropriate microscopic morphology. VSVd51 expressing human GM-CSF (VSV-hGM-CSF) was cloned from parental VSVd51 expressing GFP (VSVd51). Briefly, polymerase chain amplification was used to amplify hGM-CSF from the pUNO1-hGM-CSF plasmid (Invivogen) and the cDNA was subcloned into VSVd51 via the restriction sites XhoI/NheI. This plasmid was used to rescue a recombinant VSVd51-hGM-CSF as previously described (Alkayyal et al., 2017, Cancer Immunol Res, 5: 211-222). VSVd51 and VSVd51-mGM-CSF were obtained from the Ottawa Hospital Research Institute (Ottawa, Canada). All viruses were propagated on Vero cells and purified using Opti-Prep purification methods. Viral titers were determined by a standard plaque assay as previously published (Alkayyal et al., 2017, Cancer Immunol Res, 5: 211-222). Viral cytotoxicity was assessed on the indicated cell lines, and cell viability was carried out as described previously (Alkayyal et al., 2017, Cancer Immunol Res, 5: 211-222).
Female C57BI/6 mice (6-8 weeks old, 20-25 g) were purchased from Charles River (Quebec). Animals were housed in pathogen-free conditions at the Central Animal Facility of the Universite de Sherbrooke with access to food/water ad libitum. Animals were euthanized by cervical dislocation under anesthesia. All studies were conducted in accordance with university guidelines and the Canadian Council on Animal Care and protocols were approved by the Faculty of Medicine and Health Sciences Animal Care Committee.
For orthotopic implantation of BC cells, mice were anaesthetized and chemical lesions were induced by intravesical instillation of trypsin (Wisent) 1:1 in DMEM. During this procedure, all mice were kept under anesthesia (3% induction, 1.5% maintenance of isoflurane with 2% O2). Subsequently, 5×105 MB49 bladder tumor cells were instilled. Two days later, each group of mice received via intravesical instillation of 50 μl of 5×108 PFU of VSVd51 or VSVd51-mGM-CSF or vehicle for control groups. For the in vivo depletion of immune cell populations, 6 doses of depletion antibodies (1 dose 24 hours after tumor instillation, followed by 5 additional doses 3 days apart) were administered by intraperitoneal injection of 250 μg/dose for anti-mouse Ly6G (1A8; BioXCell); 20 μg/dose for anti-Asialo (GM1, Life Technologies) and 250 μg/dose for anti-CD8a (53-6.7, BioXCell). Bladder tumor growth was monitored bi-weekly by small animal ultrasound (Vevo3100, VisualSonics).
Following euthanasia, bladders were immediately placed in cRPMI and processed fresh using the mouse tumor dissociation kit (Miltenyi biotec). Briefly, tumors were cut into small pieces (<2 mm 3), then treated with dissociation enzymes and placed into the gentle MACS OctoDissociator (Mitenyi biotec). Following dissociation, macroscopic pieces were removed using a 70 μm nylon cell strainer. Single cell suspensions were washed twice in cRPMI and proceeded to flow cytometry acquisition and analysis as described below.
To analyze spleen, blood and tumor dissociated lymphocyte populations, an initial incubation was done in ACK lysis buffer for 5 mins to lyse red blood cells. 1×106 cells were then added to each tube. Fc block was added prior to antibody staining for 10 mins at room temperature. Samples were washed twice with flow cytometry buffer (PBS, 2% FBS, 1 mM EDTA) and acquired on a CytoFLEX 30 (Beckman Coulter). Data was analyzed with CytExpert software. For assessment of NK and T cell functionality, cells were cultured with PMA/ionomycin (Sigma) for 4 h in the presence of brefeldin A (1 μl/ml) at 37° C. After 4 h, cells were washed twice with PBS, and then stained for NK and T cell markers. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm kit, according to the manufacturer's protocol, and intracellular staining for granzyme B and IFNγ was performed. The CD107a antibody was added to cells alongside PMA/ionomycin stimulation.
For monolayer cultures, conditioned media (CM) was obtained by seeding 5×105 cells in 12-well plates in their corresponding media for 24 h followed by infection with VSVd51 and VSVd51-m/hGM-CSF at the indicated PFU for the indicated time points. For 5637 spheroids, CM was obtained by resuspending 2.5×104 5637 cells in 20 μl of Matrigel (Corning) per well of a 48-well (Thermo Fisher Scientific) plate for 6 days followed by infection with VSVd51 and VSVd51-hGM-CSF. Infected cells were harvested and processed as described above. Bioimaging was performed using an inverted microscope (Zeiss). Western blot: HMGB1 protein from CM was resolved by SDS-PAGE and transferred to Immun-Blot-PVDF membranes (BioRad) for immunoblotting. Protein expression was detected using HMGB1 primary antibodies (1:1000) and corresponding HRP-conjugated secondary antibodies (1:10000). Protein expression was visualized by chemiluminescence detection (Azure 600, Azure Biosystems). For Adenosine 5′-triphosphate (ATP) detection, the concentration of ATP in the CM was measured with the ENLITEN-ATP kit (Promega). Briefly, 100 μl of CM were transferred to 96-well opaque plates. 100 μl of reconstituted rLuciferase/Luciferin reagent was added to each well followed by measurement of luciferase activity using a luminescence microplate reader (Fusion V3.0).
Total RNA was extracted from virus-infected or mock-infected cells or organoids using Trizol (Invitrogen) according to the manufacturer's protocol. RNA was then used for reverse transcription and qPCR which was performed by the RNomics Platform at the Universite de Sherbrooke. RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Reverse transcription was performed on 1.1 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs (Roche Diagnostics), and 10 units of RNAse OUT (Invitrogen) following the manufacturer's protocol in a total volume of 10 μl. All forward and reverse primers (Supp. Table S2) were individually resuspended to 20-100 μM in Tris-EDTA buffer (IDT) and diluted as a primer pair to 1 μM in RNase DNase-free water (IDT). The amplified products were analyzed by automated chip-based microcapillary electrophoresis on Labchip GX Touch HT instruments (Perkin Elmer). QPCR reactions were performed in 10 μl in 384 well plates on a CFX-384 thermocycler (BioRad) with 5 μl of 2× PerfeCTa® SYBR® Green Supermix (Quantabio), 10 ng (3 μl) cDNA, and 200 nM final (2 μl) primer pair solutions. The following cycling conditions were used: 3 min at 95° C.; 50 cycles: 15 sec at 95° C., 30 sec at 60° C., 30 sec at 72° C. Relative expression levels were calculated using the qBASE framework and the housekeeping genes RMRP, RNU6-4P and rRNA 5.8s for human cDNA. For every qPCR run, control reactions performed in the absence of template were performed for each primer pair and these were consistently negative. Amplicon sizing and relative quantitation were performed by the manufacturer's software.
Culture supernatants were diluted 5-fold. ELISA kits (Peprotech) for detecting mouse and human GM-CSF were performed according to manufacturer's instructions.
Human monocytes were isolated from peripheral blood (Human CD14+ isolation kit, Stemcell). 5×105 monocytes were seeded in 24-well plates in complete RPMI and incubated overnight at 37° C. and 5% CO 2. 24 h later, the monocyte media was replaced with the CM of infected human cell lines. For controls, monocytes were co-cultured with recombinant human IL-10, IL-4, and TGFβ (BioBasic Inc) all at a final concentration of 20 ng/ml for differentiation to M2-like macrophages; and with LPS (50 ng/ml) (Millipore Sigma) and recombinant human IFNγ (20 ng/ml) (BioBasic Inc) for M1-like macrophages. Undifferentiated monocytes remained in complete media as M0. Following overnight incubation, cells were harvested and processed for flow cytometry as described above. 200 μl of CM were placed in the lower well of Boyden chambers, separated from the top well by a 5 μm-pore polycarbonate filters (Neuro Probe). 6×105 human PBMC was added to the top chamber, followed by incubation at 37° C., 5% CO2 for 45 mins. Next, the media in the top of the chamber was aspirated and the membrane removed with forceps. This was followed by harvesting of media in the bottom chamber and quantification of migrated cells by Trypan Blue exclusion. The cells were stained and acquired by flow cytometry as described above.
Bladder tumor tissue from patients 34 and 38 were collected after surgery (Human protocol #: 2018-2465, approved by the ethics board of CIUSSS de l'Estrie CHUS) and placed in cDMEM. Tumors were dissociated using the human tumor dissociation kit (Miltenyi biotec) according to the manufacturer's recommendations. Briefly, tumors were cut into small pieces (<2 mm 3), then treated with dissociation enzymes and placed into the gentle MACSOctoDissociator (Miltenyi biotec). Macroscopic pieces were removed using 70 μm nylon cell strainers. Tumor cells were washed twice in DMEM. Cells were viably frozen down or freshly used for downstream experiments.
Viably frozen or freshly dissociated cells (1×105) were collected by centrifugation and resuspended in 20 μl of matrigel (Corning) and plated in a prewarmed 48-well plate. When the matrigel was solidified, human bladder organoid media was added [Adv. DMEM/F-12, 100 ng/ml FGF10, 25 ng/ml FGF7, 12.5 ng/ml FGF2 (Peprotech), 1×B27 supplement (ThermoFisher), 5 μM A83-01, 1.25 mM N-acetylcysteine, and 10 mM nicotinamide (sigma)]. Human BC organoids were passaged biweekly by dissociation using TrypLE (ThermoFisher). 10 μM ROCK inhibitor (Y-27632) was added to the media after passaging to prevent cell death. Organoids were frozen in freezing media (90% FBS, 10% DMSO) and could be recovered efficiently. OV infection of organoids were performed as described for 5637 and UM-UC-3 spheroids in immunogenic cell death assays.
Immature DCs were obtained by CD14 positive selection (StemCell) according to manufacturer's guidelines from frozen human PBMCs. Sorted cells were incubated for 6 days with 500 U/ml of recombinant human IL-4 and 50 ng/ml of recombinant human GM-CSF (Bio Basic). For DC:PBMC co-culture assays, matched PBMCs were thawed incubated for 24 h with 100 U/ml of recombinant human IL-2 (Bio Basic). Following this, both DCs and lymphoid cells were incubated an additional 24 h with CM from infected autologous organoids and acquired by flow cytometry. T and NK cell functionality were assessed as described above.
All statistical analyses were conducted using Prism 7 (GraphPad). Unpaired two-tailed t tests were used for comparing uninfected or infected cells or differentially treated mice. Survival differences of tumor-bearing and treated mice were assessed using Kaplan-Meier curves and analyzed by log-rank testing. P<0.05 was considered as statistically significant.
While the description has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art to and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
The present application claims benefit of U.S. Provisional Application No. 63/209,861 filed Jun. 11, 2021 the content of which is herewith incorporated in its entirety.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/CA2022/050925 | 6/10/2022 | WO |
Number | Date | Country | |
---|---|---|---|
63209681 | Jun 2021 | US |