Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of ...irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs’ apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.
Recent data on immune evasion of new SARS-CoV-2 variants raise concerns about the efficacy of antibody-based COVID-19 therapies. Therefore, in this study the
neutralization capacity against ...SARS-CoV-2 variant B.1 and the Omicron subvariants BA.1, BA.2 and BA.5 of sera from convalescent individuals with and without boost by vaccination was assessed.
The study included 313 serum samples from 155 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=25) and with SARS-CoV-2 vaccination (n=130). We measured anti-SARS-CoV-2 antibody concentrations by serological assays (anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S) and neutralizing titers against B.1, BA.1, BA.2 and BA.5 in a pseudovirus neutralization assay. Sera of the majority of unvaccinated convalescents did not effectively neutralize Omicron sublineages BA.1, BA.2 and BA.5 (51.7%, 24.1% and 51.7%, resp.). In contrast, 99.3% of the sera of superimmunized individuals (vaccinated convalescents) neutralized the Omicron subvariants BA.1 and BA.5 and 99.6% neutralized BA.2. Neutralizing titers against B.1, BA.1, BA.2 and BA.5 were significantly higher in vaccinated compared to unvaccinated convalescents (p<0.0001) with 52.7-, 210.7-, 141.3- and 105.4-fold higher geometric mean of 50% neutralizing titers (NT50) in vaccinated compared to unvaccinated convalescents. 91.4% of the superimmunized individuals showed neutralization of BA.1, 97.2% of BA.2 and 91.5% of BA.5 with a titer ≥ 640. The increase in neutralizing titers was already achieved by one vaccination dose. Neutralizing titers were highest in the first 3 months after the last immunization event. Concentrations of anti-S antibodies in the anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S assays predicted neutralization capacity against B.1 and Omicron subvariants BA.1, BA.2 and BA.5.
These findings confirm substantial immune evasion of the Omicron sublineages, which can be overcome by vaccination of convalescents. This informs strategies for choosing of plasma donors in COVID-19 convalescent plasma programs that shall select specifically vaccinated convalescents with very high titers of anti-S antibodies.
Third-generation chimeric antigen receptor (CAR)-engineered T cells (CARTs) might improve clinical outcome of patients with B cell malignancies. This is the first report on a third-generation CART ...dose-escalating, phase-1/2 investigator-initiated trial treating adult patients with refractory and/or relapsed (r/r) acute lymphoblastic leukemia (ALL).
Thirteen patients were treated with escalating doses of CD19-directed CARTs between 1 × 10
and 50 × 10
CARTs/m
. Leukapheresis, manufacturing and administration of CARTs were performed in-house.
For all patients, CART manufacturing was feasible. None of the patients developed any grade of Immune effector cell-associated neurotoxicity syndrome (ICANS) or a higher-grade (≥ grade III) catokine release syndrome (CRS). CART expansion and long-term CART persistence were evident in the peripheral blood (PB) of evaluable patients. At end of study on day 90 after CARTs, ten patients were evaluable for response: Eight patients (80%) achieved a complete remission (CR), including five patients (50%) with minimal residual disease (MRD)-negative CR. Response and outcome were associated with the administered CART dose. At 1-year follow-up, median overall survival was not reached and progression-free survival (PFS) was 38%. Median PFS was reached on day 120. Lack of CD39-expression on memory-like T cells was more frequent in CART products of responders when compared to CART products of non-responders. After CART administration, higher CD8 + and γδ-T cell frequencies, a physiological pattern of immune cells and lower monocyte counts in the PB were associated with response.
In conclusion, third-generation CARTs were associated with promising clinical efficacy and remarkably low procedure-specific toxicity, thereby opening new therapeutic perspectives for patients with r/r ALL. Trial registration This trial was registered at www.
gov as NCT03676504.
The Epstein-Barr virus (EBV) induces B-cell proliferation with high efficiency through expression of latent proteins and microRNAs. This process takes place in vivo soon after infection, presumably ...to expand the virus reservoir, but can also induce pathologies, e.g. an infectious mononucleosis (IM) syndrome after primary infection or a B-cell lymphoproliferation in immunosuppressed individuals. In this paper, we investigated the growth characteristics of EBV-infected B-cells isolated from transplant recipients or patients with IM. We found that these cells grew and withstood apoptosis at highly variable rates, suggesting that the expansion rate of the infected B-cells widely varies between individuals, thereby influencing the size of the B-cell reservoir and the ability to form tumors in infected individuals. All viruses investigated were type 1 and genetically close to western strains. EBV-infected B-cells expressed the transforming EBV latent genes and microRNAs (miRNAs) at variable levels. We found that the B-cell growth rates positively correlated with the BHRF1 miRNA levels. Comparative studies showed that infected B-cells derived from transplant recipients with iEBVL on average expressed higher levels of EBV miR-BHRF1 miRNAs and grew more rapidly than B-cells from IM patients, suggesting infection by more transforming viruses. Altogether, these findings suggest that EBV infection has a highly variable impact on the B-cell compartment that probably reflects the genetic diversity of both the virus and the host. It also demonstrates the unexpected finding that B-cells from different individuals can grow at different speed under the influence of the same virus infection.
High-dose (HD) chemotherapy followed by autologous blood stem-cell transplantation (ASCT) is the standard treatment for multiple myeloma (MM) patients. However, the collection of sufficient ...peripheral blood stem cell (PBSC) grafts can be challenging, and the question arises whether reinfusion of low-dose grafts will lead to a hematopoietic recovery.
The hematopoietic recovery of 148 MM patients who underwent HD melphalan chemotherapy and received PBSC transplants with varying CD34+ cells doses (3-4 × 10
n = 86, 2-2.5 × 10
n = 53, < 2 × 10
n = 9 per kg body weight bw) was analyzed in this retrospective single-center study.
All patients reached hematopoietic reconstitution, even those who received < 2 × 10
CD34+ cells/kg bw. 62 (42%) patients received granulocyte-colony-stimulating factor (G-CSF). The median duration to leukocyte recovery ≥1.0 × 10
/L was 12 days in every group. The median duration to platelet recovery ≥20 × 10
/L was 11, 13 and 13 days, respectively. In the multivariate analysis, a low number of reinfused CD34+ cells was associated with prolonged time until leukocyte reconstitution (p = 0.010, HR 0.607) and platelet recovery (p < 0.001, HR 0.438). G-CSF support significantly accelerated leukocyte (p < 0.001, HR 16.742) but not platelet reconstitution.
In conclusion, reinfusion of low- and even very-low-dose PBSC grafts leads to sufficient hematopoietic reconstitution. No severe adverse events were observed during or after HD chemotherapy and ASCT in the analyzed cohort. While the impact of CD34+ cell dose is marginal, G-CSF significantly accelerates the leukocyte recovery.
Hyperthermia demonstrated clinical efficacy in multimodal cancer treatment. Multipotent mesenchymal stromal cells (MSCs) as part of the tumor-supporting stroma modulate tumor response and tissue ...regeneration after hyperthermia. We aimed to investigate the effects of hyperthermia on the survival, stem cell characteristics and heat shock expression of human MSCs.
Human MSCs and normal human dermal fibroblasts (NHDFs) were exposed to temperatures between 37 °C and 44 °C for 60 min, and hyperthermic sensitivity was examined by clonogenicity, proliferation and viability assays. The influence of 42 °C hyperthermia on the MSCs' adhesion potential, migratory capacity, surface marker expression and multi-lineage differentiation capability was investigated. Cell cycle distribution, apoptosis and senescence after 42 °C hyperthermia were determined by flow cytometry and β-galactosidase staining. Heat shock protein expression was determined by Western Blots.
MSCs exhibited decreased clonogenic survival after 40 °C and 42 °C hyperthermia compared to NHDFs, while proliferative activity and viability were comparable after hyperthermia up to 44 °C. MSC adhesion was reduced after 42 °C hyperthermia, while the characteristic surface marker expression and the migratory ability remained unaffected in 42 °C hyperthermia-exposed MSCs. 42 °C hyperthermia diminished the adipogenic differential potential of all tested MSC samples. A pronounced G2/M arrest was found after 42 °C hyperthermia and was associated with increased apoptosis and senescence levels in MSCs. MSCs exhibited slightly lower heat shock protein levels compared to NHDFs.
Human MSCs exhibit a thermosensitive phenotype which reduced the multipotent cells' regenerative abilities, resulting in impaired tissue regeneration after hyperthermia treatment or thermal injuries. On the other hand, tumor-associated MSCs may be efficiently targeted by hyperthermia treatment.
Robust and reliable in vitro and in vivo models of primary cells are necessary to study the pathomechanisms of Myelodysplastic Neoplasms (MDS) and identify novel therapeutic strategies. MDS-derived ...hematopoietic stem and progenitor cells (HSPCs) are reliant on the support of bone marrow (BM) derived mesenchymal stroma cells (MSCs). Therefore, isolation and expansion of MCSs are essential for successfully modeling this disease. For the clinical use of healthy MSCs isolated from human BM, umbilical cord blood or adipose tissue, several studies showed that xeno-free (XF) culture conditions resulted in superior growth kinetics compared to MSCs cultured in the presence of fetal bovine serum (FBS). In this present study, we investigate, whether the replacement of a commercially available MSC expansion medium containing FBS with a XF medium is beneficial for the expansion of MSCs derived from BM of MDS patients which are often difficult to cultivate.
MSCs isolated from BM of MDS patients were cultured and expanded in MSC expansion medium with FBS or XF supplement. Subsequently, the impact of culture media on growth kinetics, morphology, immunophenotype, clonogenic potential, differentiation capacity, gene expression profiles and ability to engraft in immunodeficient mouse models was evaluated.
Significant higher cell numbers with an increase in clonogenic potential were observed during culture of MDS MSCs with XF medium compared to medium containing FBS. Differential gene expression showed an increase in transcripts associated with MSC stemness after expansion with XF. Furthermore, immunophenotypes of the MSCs and their ability to differentiate into osteoblasts, adipocytes or chondroblasts remained stable. MSCs expanded with XF media were similarly supportive for creating MDS xenografts in vivo as MSCs expanded with FBS.
Our data indicate that with XF media, higher cell numbers of MDS MSCs can be obtained with overall improved characteristics in in vitro and in vivo experimental models.
The occurrence of skeletal metastases in cancer, e.g. breast cancer (BC), deteriorates patient life expectancy and quality-of-life. Current treatment options against tumor-associated bone disease are ...limited to anti-resorptive therapies and aimed towards palliation. There remains a lack of therapeutic approaches, which reverse or even prevent the development of bone metastases. Recent studies demonstrate that not only osteoclasts (OCs), but also osteoblasts (OBs) play a central role in the pathogenesis of skeletal metastases, partly by producing hepatocyte growth factor (HGF), which promotes tumor cell migration and seeding into the bone. OBs consist of a heterogeneous cell pool with respect to their maturation stage and function. Recent studies highlight the critical role of pre-OBs in hematopoiesis. Whether the development of bone metastases can be attributed to a particular OB maturation stage is currently unknown.
Pre-OBs were generated from healthy donor (HD)-derived bone marrow stromal cells (BMSC) as well as the BMSC line KM105 and defined as ALPlow OPNlow RUNX2high OSX high CD166high. Conditioned media (CM) of pre-OBs, but not of undifferentiated cells or mature OBs, enhanced migration of metastatic BC cells. Importantly, HGF mRNA was significantly up-regulated in pre-OBs versus mature OBs, and CM of pre-OBs activated the MET signaling pathway. Highlighting a key role for HGF, CM from HGF-negative pre-OBs derived from the BMSC line HS27A did not support migration of BC cells. Genetically (siMET) or pharmacologically (INCB28060) targeting MET inhibited both HGF- and pre-OB CM- mediated BC cell migration.
Our data demonstrate for the first time a role for pre-OBs in mediating HGF/MET- dependent migration of BC cells and strongly support the clinical evaluation of INCB28060 and other MET inhibitors to limit and/or prevent BC-associated bone metastases.
Mesenchymal stem cells (MSCs) participate in the regeneration of tissue lesions induced by antimetabolite chemotherapy; however, the influence of this class of anti-cancer compounds on the stem cells ...remains largely unknown.
The survival of MSCs after exposure to 5-fluorouracil (5-FU) and gemcitabine was measured by viability and clonogenic assays. MSC morphology, surface marker expression, adhesion potential, cellular velocity and differentiation potential were determined after antimetabolite treatment. Cell cycle distribution and apoptosis were assessed using flow cytometry, and senescence induction was evaluated by beta-galactosidase staining. Gene expression arrays were used to analyze the expression of enzymes involved in DNA metabolism and multidrug resistance.
Here, we show that human primary bone marrow MSCs are relatively resistant to treatment with the widely used antimetabolite drugs 5-FU and gemcitabine. The stem cells were able to largely retain their functional abilities and defining stem cell traits after antimetabolite exposure. MSCs surface markers were found stably expressed, and the characteristic multi-lineage differentiation potential was maintained irrespective of 5-FU or gemcitabine treatment. High expression levels of enzymes involved in DNA metabolism and multidrug resistance transporters may contribute to the resistance to antimetabolite chemotherapy.
The observed resistance and functional integrity may form the basis for further investigations of MSCs as a potential therapy for antimetabolite-induced tissue damage.
•Human mesenchymal stem cells are relatively resistant to antimetabolite treatment.•MSCs maintain their defining characteristics after antimetabolite treatment.•High expression of enzymes for DNA metabolism and multidrug resistance transporters may explain antimetabolite resistance.•MSCs may serve to attenuate antimetabolite-induced tissue damage.
Using planar lipid membranes with precisely defined concentrations of specific ligands, we have determined the binding strength between human hematopoietic stem cells (HSC) and the bone marrow niche. ...The relative significance of HSC adhesion to the surrogate niche models via SDF1α-CXCR4 or N-cadherin axes was quantified by (a) the fraction of adherent cells, (b) the area of tight adhesion, and (c) the critical pressure for cell detachment. We have demonstrated that the binding of HSC to the niche model is a cooperative process, and the adhesion mediated by the CXCR4- SDF1α axis is stronger than that by homophilic N-cadherin binding. The statistical image analysis of stochastic morphological dynamics unraveled that HSC dissipated energy by undergoing oscillatory deformation. The combination of an in vitro niche model and novel physical tools has enabled us to quantitatively determine the relative significance of binding mechanisms between normal HSC versus leukemia blasts to the bone marrow niche.
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