MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option.

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSCenr -EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr -EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr -EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr -EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSCenr -EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSCenr -EXOs disrupted spheroids' structure. Thus, CSCenr -EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.

Enhancement of anti-inflammatory and immunomodulatory effects of adipose-derived human mesenchymal stem cells by making uniform spheroid on the new nano-patterned plates.

Human mesenchymal stem cells (MSCs) are known to have anti-inflammatory and immunomodulatory functions; thus, several MSC products have been applied as cell therapy in clinical trials worldwide. Recent studies have demonstrated that MSC spheroids have superior anti-inflammatory and immunomodulatory functions to a single cell suspension. Current methods to prepare MSC spheroids include hanging drop, concave microwell aggregation, spinner flask, and gravity circulation. However, all these methods have limitations such as low scalability, easy cell clumping, low viability, and irregular size distribution. Here, we present a nano-patterned culture plasticware named PAMcell™ 3D plate to overcome these limitations. Nano-sized silica particles (700 nm) coated with RGD peptide were arrayed into fusiform onto the PLGA film. This uniform array enabled the seeded MSCs to grow only on the silica particles, forming uniform-sized semi-spheroids within 48 h. These MSC spheroids have been shown to have enhanced stemness, anti-inflammatory, and immunomodulatory functions, as revealed by the increased expression of stem cell markers (Oct4, Sox2, and Nanog), anti-inflammatory (IL-10, TSG6, and IDO), and immunomodulatory molecules (HGF, VEGF, CXCR4) both at mRNA and protein expression levels. Furthermore, these MSC spheroids demonstrated an increased palliative effect on glycemic control in a multiple low-dose streptozotocin-induced diabetes model compared with the same number of MSC single cell suspensions. Taken together, this study presents a new method to produce uniform-sized MSC spheroids with enhanced anti-inflammatory and immunomodulatory functions in vitro and in vivo.

Reversing Hypoxia with PLGA-Encapsulated Manganese Dioxide Nanoparticles Improves Natural Killer Cell Response to Tumor Spheroids.

The adoptive transfer of natural killer (NK) cells, which can recognize and obliterate cancer cells, provides a practical alternative to current treatment modalities to improve cancer patients' survival. However, translating NK cell therapies to treat solid tumors has proven challenging due to the tumor microenvironment (TME). Hypoxia in the TME induces immunosuppression that inhibits the cytotoxic function of NK cells. Thus, reversing hypoxia-induced immunosuppression is critical for effective adoptive NK cell immunotherapy. In this study, we use manganese dioxide nanoparticles (MnO2 NPs) to catalyze the degradation of tumor-produced hydrogen peroxide, thereby generating oxygen. For improved biocompatibility and modulation of oxygen production, the MnO2 NPs were encapsulated into poly(lactic-co-glycolic) to produce particles that are 116 nm in size and with a ζ-potential of +17 mV (PLGA-MnO2 NPs). The PLGA-MnO2 NPs showed first-order oxygen production and sustained high oxygen tension compared to equivalent amounts of bare MnO2 NPs in the presence of H2O2. The PLGA-MnO2 NPs were biocompatible, reduced hypoxia after penetration into the core of cancer spheroids, and decreased hypoxia-induced factor 1 α expression. Reducing hypoxia in the spheroid resulted in a decrease in the potent immunosuppressors, adenosine, and lactate, which was confirmed by electrospray ionization mass spectroscopy (ESI-MS). ESI-MS also showed a change in the metabolism of the amino acids aspartate, glutamine, and glutamate after hypoxia reduction in the cancer cells. Notably, the spheroids' microenvironment changes enhanced NK cells' cytotoxicity, which obliterated the spheroids. These results demonstrate that reducing hypoxia-induced immunosuppression in tumors is a potent strategy to increase the potency of cytotoxic immune cells in the TME. The developed NPs are promising new tools to improve adoptive NK cell therapy.

CD44+CD24-/low sphere-forming cells of EBV-associated gastric carcinomas show immunosuppressive effects and induce Tregs partially through production of PGE2.

EBV-associated gastric carcinoma (EBVaGC) is accompanied by massive lymphocyte infiltration, but therapy resistance and tumor progression still occur in patients with EBVaGC. Cancer stem cells (CSCs) are reported to possess immunomodulatory ability that allows them to resist immune-mediated rejection for many tumor types. However, whether and how CSCs in EBVaGC exhibit immunosuppression has not yet been elucidated. We isolated CSC-like sphere-forming cells (SFCs) from EBVaGC cell line SNU-719 using the cancer sphere method. We validated their CSC-associated properties in the expression of the epithelial-mesenchymal transition (EMT)-related genes, the ability to form colonies, and resistance to chemotherapy drug-induced apoptosis and explored their immunomodulatory ability using the coculture system with PBMC (peripheral blood mononuclear cell). These CSC-like SFCs were CD44+CD24-/low and were more tumorigenic than the parental SNU-719 cells in the xenograft mouse model. Remarkably, in the tumor-PBMC co-culturing experiments, these EBVaGC SFCs demonstrated profound immunosuppression by inhibiting the proliferation of PBMCs and T cell activation as well as inducing the generation of regulatory T cells (Tregs). Furthermore, the induction of Tregs was partially dependent on prostaglandin E2 (PGE2) produced from SFCs. Moreover, the presence of high CD44+CD24-/low cells in tumor tissues predicted a decreased disease-free survival in patients with EBVaGC. Our study collectively confirmed the existence and immune resistance of CSCs in EBVaGC and offers new insights into the development of novel anti-EBVaGC strategies by targeting CSCs.

A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication.

Cancer-derived extracellular vesicles (EVs) have emerged as important mediators of tumour-host interactions, and they have been shown to exert various functional effects in immune cells. In most of the studies on human immune cells, EVs have been isolated from cancer cell culture medium or patients' body fluids and added to the immune cell cultures. In such a setting, the physiological relevance of the chosen EV concentration is unknown and the EV isolation method and the timing of EV administration may bias the results. In the current study we aimed to develop an experimental cell culture model to study EV-mediated effects in human T and B cells at conditions mimicking the tumour microenvironment. We constructed a human prostate cancer cell line PC3 producing GFP-tagged EVs (PC3-CD63-GFP cells) and developed a 3D heterotypic spheroid model composed of PC3-CD63-GFP cells and human peripheral blood mononuclear cells (PBMCs). The transfer of GFP-tagged EVs from PC3-CD63-GFP cells to the lymphocytes was analysed by flow cytometry and fluorescence imaging. The endocytic pathway was investigated using three endocytosis inhibitors. Our results showed that GFP-tagged EVs interacted with a large fraction of B cells, however, the majority of EVs were not internalised by B cells but rather remained bound at the cell surface. T cell subsets differed in their ability to interact with the EVs - 15.7-24.1% of the total CD3+ T cell population interacted with GFP-tagged EVs, while only 0.3-5.8% of CD8+ T were GFP positive. Furthermore, a fraction of EVs were internalised in CD3+ T cells via macropinocytosis. Taken together, the heterotypic PC3-CD63-GFP and PBMC spheroid model provides the opportunity to study the interactions and functional effects of cancer-derived EVs in human immune cells at conditions mimicking the tumour microenvironment.

Intracoronary allogeneic cardiosphere-derived stem cells are safe for use in dogs with dilated cardiomyopathy.

Cardiosphere-derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non-ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c-kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells.

In vitro Monocyte IL-6 Secretion Levels Following Stimulation with Autologous Spheroids Derived from Tumour or Benign Mucosa Predict Long-term Survival in Head and Neck Squamous Cell Carcinoma Patients.

MCP-1/IL-6 in vitro monocyte secretion upon coculture with autologous fragment spheroids was studied in relation to patient 5- and 10-year overall survival rates in head and neck squamous cell carcinoma (HNSCC) patients (n = 65) diagnosed between 1998 and 2005, nine of whom had an human papilloma virus (HPV) tumour infection. The spheroids were harvested from malignant or benign tissue during primary surgery. Two weeks following surgery, freshly isolated autologous monocytes and benign or malignant spheroids were cocultured 24 h in vitro. The IL-6 secretion was expressed as a fraction of the lipopolysaccharide (LPS) response from the same batch of monocytes. HPV status was obtained by employing PCR analyses of primary diagnostic blocks. IL-6/MCP-1 response levels were not found to be dependent on HPV infection status. MCP-1 secretion did not predict prognosis, nor did in vitro IL-6 monocyte background or LPS-stimulated IL-6 secretion. At 5-year observation, dichotomized IL-6 levels following monocyte coculture, with both malignant and benign spheroids, showed a strong trend towards predicting survival, that is a low monocyte malignant coculture response showed a survival of 31 ± 17 versus 58 ± 17% with a high such response (P = 0.057). When studying monocyte IL-6 coculture responses evaluating benign and malignant spheroid results statistically together, a prediction of survival up to 10 years was found (hazard ratio = 0.48; confidence interval = 0.24-0.96; P < 0.05) with double low IL-6 responses. This survival prediction was also present after an adjustment for HPV tumour infection status. In conclusion, monocyte IL-6 in vitro secretion in cocultures with autologous spheroids/serum from HNSCCs predicted 5- and 10-year survivals, both with and without tumour HPV tumour adjustment.

Epithelial ovarian cancer stem-like cells expressing α-gal epitopes increase the immunogenicity of tumor associated antigens.

BACKGROUND: As ovarian cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and chemo-resistance, new stratagems that selectively target ovarian CSCs are critically significant. Our previous work have demonstrated that ovarian cancer spheroid cells are tumorigenic and chemo-resistant, and have the properties of ovarian CSCs. Herein, we hypothesized that expressing α-gal epitopes on ovarian spheroid cells may help eliminate CSCs and improve the outcome of therapeutic intervention for ovarian cancer patients. METHODS: Lentivirus-mediated transfer of a pig α(1,3)galactosyltransferase [α1,3GT] enzyme gene into human ovarian cell line SKOV3 cells formed α-gal epitope-expressing cells (SKOV3-gal cells), and then these cells were maintained in a serum-free culture system to form SKOV3-gal spheroid cells. Efficacy of this cell vaccine was demonstrated in α1,3GT knockout mice (α1,3GT KO mice). RESULTS: The antibody titers to α-gal epitopes measured by ELISA were significantly increased in α1,3GT KO mice after immunization with SKOV3-gal spheroid cells. Furthermore, compared with the non-immunized KO mice, the SKOV3 tumors grafted under renal capsules of KO mice immunized with SKOV3-gal spheroid cells grew slower and began to shrink on day 12. Western blot analysis also showed that immunized KO mice can produce effective antibody against certain tumor associated antigens (TAAs) derived from both SKOV3 cells and SKOV3 spheroid cells. The TAAs were further investigated by mass spectrometry and RNA interference (RNAi) technology. The results suggested that antibodies responding to protein c-erbB-2 may be raised in the sera of the mice after immunization with SKOV3-gal spheroid cells. Ultimately, vaccination with SKOV3-gal spheroid cells induced more CD3+CD4+T cells in the spleen of immunized mice than non-immunized KO mice. CONCLUSIONS: The results suggest that vaccination using ovarian cancer stem-like cells engineered to express α-gal epitopes may be a novel strategy for treatment of ovarian cancer.

[3D tumor spheroids promote activation, expansion, and anti-tumor effects of tumor-infiltrating lymphocytes in vitro].

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3+/CD8+ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3+/CD8+ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3+/CD8+ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells.

Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck(-/-) macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.

A human NK cell activation/inhibition threshold allows small changes in the target cell surface phenotype to dramatically alter susceptibility to NK cells.

NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing's sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing's sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.

[Zajdela hepatoma cells cultured in vitro].

Two continuous cell lines, monolayer (ZH-ad) and suspension (ZH-fl), which doffer in the level of cell differentiation and tumorigenicity, were obtained from explants of ascitic Zajdela hepatoma. Using tumor-specific rabbit immune serum tumor-associated antigens were detected in the fraction of plasma membrane of ZH-fl cells, synthesis of that was reduced or suppressed in ZH-ad cells. Intraperitoneal injection of ZH-fl cells as few as 0.5 - 10(6) cells/rat was enough for development of ascites tumor, while minimal dose for ZH-ad cells increase to 20 - 10(6) cells. Three types of generated clones were revealed by means of clonogenic analysis of ZH-fl cells: nonadhesive sphere colonies and two types of monolayer clones ranging in proliferative potential, shape of colonies and cell composition. When the spherical colonies had reached a critical size they disintegrated with dissociation of single cells or islets of various sizes, part of which attached to the plate to form a monolayer. Three clonal cell lines were obtained: IC - as a result of expansion of sphere clone cells, 4G and 10E - from monolayer clones. Our results demonstrate a clonogenic origin of multicellular islets of ascetic hepatoma Zajdela.

The ganglioside antigen G(D2) is surface-expressed in Ewing sarcoma and allows for MHC-independent immune targeting.

BACKGROUND: Novel treatment strategies are needed to cure disseminated Ewing sarcoma. Primitive neuroectodermal features and a mesenchymal stem cell origin are both compatible with aberrant expression of the ganglioside antigen G(D2) and led us to explore G(D2) immune targeting in this cancer. METHODS: We investigated G(D2) expression in Ewing sarcoma by immunofluorescence staining. We then assessed the antitumour activity of T cells expressing a chimeric antigen receptor specific for G(D2) against Ewing sarcoma in vitro and in vivo. RESULTS: Surface G(D2) was detected in 10 out of 10 Ewing sarcoma cell lines and 3 out of 3 primary cell cultures. Moreover, diagnostic biopsies from 12 of 14 patients had uniform G(D2) expression. T cells specifically modified to express the G(D2)-specific chimeric receptor 14. G2a-28ζ efficiently interacted with Ewing sarcoma cells, resulting in antigen-specific secretion of cytokines. Moreover, chimeric receptor gene-modified T cells from healthy donors and from a patient exerted potent, G(D2)-specific cytolytic responses to allogeneic and autologous Ewing sarcoma, including tumour cells grown as multicellular, anchorage-independent spheres. G(D2)-specific T cells further had activity against Ewing sarcoma xenografts. CONCLUSION: G(D2) surface expression is a characteristic of Ewing sarcomas and provides a suitable target antigen for immunotherapeutic strategies to eradicate micrometastatic cells and prevent relapse in high-risk disease.

T cells sensitized with breast tumor progenitor cell vaccine have therapeutic activity against spontaneous HER2/neu tumors.

Cancer progenitor cells are critical for tumor initiation and recurrence so they are an important therapeutic target. We tested whether T cells could recognize tumor antigens expressed by breast cancer progenitor cells and acquire therapeutic activity against established metastases or delay onset of spontaneous tumors. Breast tumors were derived from HER2/neu transgenic mice and propagated in vitro under conditions that selected progenitor cells which were then used as an irradiated whole cell vaccine. A minor subset of recently sensitized T cells was isolated from vaccine-draining lymph nodes then activated in vitro to achieve numerical expansion. We show that the tumor progenitor cell vaccines reversed tolerance to a known HER2/neu epitope, otherwise inhibited by Treg cells. Additional shared tumor antigens were recognized because a Neuneg subclone also induced a Th1 type immune response against breast tumors. Adoptive transfer of in vitro activated lymph node T cells-mediated regression of established metastases from multiple independently derived breast tumor lines. Moreover, adoptive transfer of effector T cells into Neu-tolerant mice, months before the onset of spontaneous tumors, significantly postponed tumor development. Interestingly, T-cell-mediated lysis of metastases stimulated an IgG response to HER2/neu as well as other shared antigens. In summary, tumor progenitor cells contain shared antigens which can lead to a cross-protective T-cell response. Moreover, antigens acquired during immune-mediated tumor destruction are presented in a manner conducive to reversal of tolerance and Ig class switching. These complementary effector mechanisms might augment therapy by eliminating refractory breast cancer stem cells.

Effect of CD147 monoclonal antibody on paclitaxel resistance in HCE1 multicellular spheroids.

OBJECTIVE: To investigate the effect of CD147 monoclonal antibody (mAb) on the natural resistance to paclitaxel (TAX) in the human cervical cancer line (HCE1) multicellular spheroid (HCE1/MCS) model and if CD147 mAb can reverse the HCE1/MCS resistance to TAX. METHODS: HCE1/MCS was obtained by liquid overlay and rotating technique. HCE1/MCS morphological changes were observed before or after the interference of CD147 mAb. The effects of TAX on HCE1/MCS (including inhibition ratio, IC50 and index of multicellular resistance) before or after CD147 mAb treatment were determined by the method of WST-1 and the inhibition ratio curve was mapped. Cell cycle and apoptosis were detected by flow cytometer (FCM). The expression of CD147 and P-gp of both HCE1/MC and HCE1/MCS was detected by immunocytochemistry. RESULTS: HCE1/MCS was established successfully. CD147 mAb could inhibit HCE1/MCS from forming spheroids. CD147 mAb could enhance the sensitivity of HCE1/MCS to TAX. IC50 in different concentrations of CD147 mAb (5,10,20 µg/mL) HCE1/MCS group were (40.31 ± 3.73), (32.43 ± 1.56), and (30.69 ± 1.01) µg/mL. CD147 mAb resulted in G1/G0 arrest in HCE1/MCS. CD147 mAb of low concentrations (0-10 µg/mL) caused a dose-dependent inhibition of HCE1/MCS (P<0.05). Combined with TAX, CD147 mAb could also induce HCE1/MCS cell cycle arrest in both G1/S and G2/M stage. The expression of CD147 and P-gp was consistent in HCE1/MCS groups. CONCLUSION: CD147 plays an important role in muliticellular resistance of cervical cancer and inhibition of CD147 can synergistically reverse the multicellular drug resistance (MCR) in cervical cancer. The MCR of HCE1/MCS mediated by CD147 is related to P-gp.

Immunomodulation effect of mesenchymal stem cells in islet transplantation.

Mesenchymal stem cells (MSCs) therapy has brought a great enthusiasm to the treatment of various immune disorders, tissue regeneration and transplantation therapy. MSCs are being extensively investigated for their immunomodulatory actions. MSCs can deliver immunomodulatory signals to inhibit allogeneic T cell immune responses by downregulating pro-inflammatory cytokines and increasing regulatory cytokines and growth factors. Islet transplantation is a therapeutic alternative to the insulin therapy for the treatment of type 1 diabetes mellitus (T1DM). However, the acute loss of islets due to the lack of vasculature and hypoxic milieu in the immediate post-transplantation period may lead to treatment failure. Moreover, despite the use of potent immunosuppressive drugs, graft failure persists because of immunological rejection. Many in vitro and in vivo researches have demonstrated the multipotency of MSCs as a mediator of immunomodulation and a great approach for enhancement of islet engraftment. MSCs can interact with immune cells of the innate and adaptive immune systems via direct cell-cell contact or through secretomes containing numerous soluble growth and immunomodulatory factors or mitochondrial transfer. This review highlights the interactions between MSCs and different immune cells to mediate immunomodulatory functions along with the importance of MSCs therapy for the successful islet transplantation.

Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade.

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.

Establishment of a Patient-Derived, Magnetic Levitation-Based, Three-Dimensional Spheroid Granuloma Model for Human Tuberculosis.

Tuberculous granulomas that develop in response to Mycobacterium tuberculosis (M. tuberculosis) infection are highly dynamic entities shaped by the host immune response and disease kinetics. Within this microenvironment, immune cell recruitment, polarization, and activation are driven not only by coexisting cell types and multicellular interactions but also by M. tuberculosis-mediated changes involving metabolic heterogeneity, epigenetic reprogramming, and rewiring of the transcriptional landscape of host cells. There is an increased appreciation of the in vivo complexity, versatility, and heterogeneity of the cellular compartment that constitutes the tuberculosis (TB) granuloma and the difficulty in translating findings from animal models to human disease. Here, we describe a novel biomimetic in vitro three-dimensional (3D) human lung spheroid granuloma model, resembling early "innate" and "adaptive" stages of the TB granuloma spectrum, and present results of histological architecture, host transcriptional characterization, mycobacteriological features, cytokine profiles, and spatial distribution of key immune cells. A range of manipulations of immune cell populations in these spheroid granulomas will allow the study of host/pathogen pathways involved in the outcome of infection, as well as pharmacological interventions. IMPORTANCE TB is a highly infectious disease, with granulomas as its hallmark. Granulomas play an important role in the control of M. tuberculosis infection and as such are crucial indicators for our understanding of host resistance to TB. Correlates of risk and protection to M. tuberculosis are still elusive, and the granuloma provides the perfect environment in which to study the immune response to infection and broaden our understanding thereof; however, human granulomas are difficult to obtain, and animal models are costly and do not always faithfully mimic human immunity. In fact, most TB research is conducted in vitro on immortalized or primary immune cells and cultured in two dimensions on flat, rigid plastic, which does not reflect in vivo characteristics. We have therefore conceived a 3D, human in vitro spheroid granuloma model which allows researchers to study features of granuloma-forming diseases in a 3D structural environment resembling in vivo granuloma architecture and cellular orientation.