A Label-Free Cell Sorting Approach to Highlight the Impact of Intratumoral Cellular Heterogeneity and Cancer Stem Cells on Response to Therapies.

Cancer stem cells play a crucial role in tumor initiation, metastasis, and resistance to treatment. Cellular heterogeneity and plasticity complicate the isolation of cancer stem cells. The impact of intra-tumor cellular heterogeneity using a label-free approach remains understudied in the context of treatment resistance. Here, we use the sedimentation field-flow fractionation technique to separate, without labeling, cell subpopulations of colorectal cancer cell lines and primary cultures according to their biophysical properties. One of the three sorted cell subpopulations exhibits characteristics of cancer stem cells, including high tumorigenicity in vivo and a higher frequency of tumor-initiating cells compared to the other subpopulations. Due to its chemoresistance, two- and three-dimensional in vitro chemosensitivity assays highlight the therapeutic relevance of this cancer stem cell subpopulation. Thus, our results reveal the major implication of intra-tumor cellular heterogeneity, including cancer stem cells in treatment resistance, thanks to our label-free cell sorting approach. This approach enables-by breaking down the tumor-the study the individualized response of each sorted tumor cell subpopulation and to identify chemoresistance, thus offering new perspectives for personalized therapy.

Preclinical study of 212Pb alpha-radioimmunotherapy targeting CD20 in non-Hodgkin lymphoma.

BACKGROUND: Despite therapeutic advances, Non-Hodgkin lymphoma (NHL) relapses can occur. The development of radioimmunotherapy (RIT) with α-emitters is an attractive alternative. In this study, we investigated the potential of α-RIT in conjunction with 212Pb-rituximab for the treatment of NHL. METHODS: EL4-hCD20-Luc cells (mouse lymphoma cell line) were used for in vitro and in vivo studies. Biodistribution and efficacy studies were performed on C57BL/6 mice injected intravenously with 25 × 103 cells. RESULTS: 212Pb-rituximab (0.925-7.4 kBq/mL) inhibit proliferation of EL4-hCD20-Luc cells in vitro. Biodistribution of 203/212Pb-rituximab in mice showed a significant tumour uptake and suggested that the liver, spleen, and kidneys were the organs at risk. For efficacy studies, mice were treated at either 11 days (early stage) or 20-30 days after injection of tumour cells (late stage). Treatment with 277.5 kBq 212Pb-rituximab significantly prolonged survival. Even at an advanced tumour stage, significant tumour regression occurred, with an increase in the median survival time to 28 days, compared with 9 days in the controls. CONCLUSIONS: These results show the efficacy of 212Pb-rituximab in a murine syngeneic lymphoma model, in terms of significant tumour regression and increased survival, thereby highlighting the potency of α-RIT for the treatment of NHL.

212Pb α-Radioimmunotherapy Targeting CD38 in Multiple Myeloma: A Preclinical Study.

Multiple myeloma (MM) is a plasma cell cancer and represents the second most frequent hematologic malignancy. Despite new treatments and protocols, including high-dose chemotherapy associated with autologous stem cell transplantation, the prognosis of MM patients is still poor. α-radioimmunotherapy (α-RIT) represents an attractive treatment strategy because of the high-linear-energy transfer and short pathlength of α-radiation in tissues, resulting in high tumor cell killing and low toxicity to surrounding tissues. In this study, we investigated the potential of α-RIT with 212Pb-daratumumab (anti-hCD38), in both in vitro and in vivo models, as well as an antimouse CD38 antibody using in vivo models. Methods: Inhibition of cell proliferation after incubation of the RPMI8226 cell line with an increasing activity (0.185-3.7 kBq/mL) of 212Pb-isotypic control or 212Pb-daratumumab was evaluated. Biodistribution was performed in vivo by SPECT/CT imaging and after death. Dose-range-finding and acute toxicity studies were conducted. Because daratumumab does not bind the murine CD38, biodistribution and dose-range finding were also determined using an antimurine CD38 antibody. To evaluate the in vivo efficacy of 212Pb-daratumumab, mice were engrafted subcutaneously with 5 × 106 RPMI8226 cells. Mice were treated 13 d after engraftment with an intravenous injection of 212Pb-daratumumab or control solution. Therapeutic efficacy was monitored by tumor volume measurements and overall survival. Results: Significant inhibition of proliferation of the human myeloma RPMI8226 cell line was observed after 3 d of incubation with 212Pb-daratumumab, compared with 212Pb-isotypic control or cold antibodies. Biodistribution studies showed a specific tumoral accumulation of daratumumab. No toxicity was observed with 212Pb-daratumumab up to 370 kBq because of lack of cross-reactivity. Nevertheless, acute toxicity experiments with 212Pb-anti-mCD38 established a toxic activity of 277.5 kBq. To remain within realistically safe treatment activities for efficacy studies, mice were treated with 185 kBq or 277.5 kBq of 212Pb-daratumumab. Marked tumor growth inhibition compared with controls was observed, with a median survival of 55 d for 277.5 kBq of 212Pb-daratumumab instead of 11 d for phosphate-buffered saline. Conclusion: These results showed 212Pb-daratumumab to have efficacy in xenografted mice, with significant tumor regression and increased survival. This study highlights the potency of α-RIT in MM treatment.