Autologous stem cell transplantation (ASCT) is an effective treatment strategy for multiple myeloma (MM) but isolating enough hematopoietic stem and progenitor cells (HSPCs) for transplantation can be challenging. The selective CXCR4 inhibitor motixafortide has been shown to be a safe and efficacious HSPC mobilization agent in a phase 2 trial when paired with G-CSF. Crees et al. (2023) report the results of the phase 3 trial, in which they used the ZE5 Cell Analyzer to explore motixafortide’s mobilizing effects.

Use flow cytometry
for cancer therapy development.

Learn More

MM makes up about 10% of all hematological malignancies. In the U.S., more than 32,000 new diagnoses are made each year with approximately 13,000 of these individuals dying of the disease (Albagoush et al. 2023, Rajkumar 2022). MM is a type of cancer that affects the antibody-producing plasma cells in the bone marrow, causing uncontrolled proliferation and overproduction of antibodies. This, in turn, can lead to neurological defects caused by hyperviscosity of the blood, excessive bleeding because of platelet dysfunction, kidney problems resulting in renal failure, and lytic bone lesions due to the simultaneous activation of bone-resorbing osteoclasts and suppression of bone-forming osteoblasts.

Although MM is not currently curable, several treatment options exist which can encourage remission. In newly diagnosed patients, high-dose chemotherapy can be used to kill myeloma cells, this may be followed by autologous stem cell transplantation (ASCT) to replenish the bone marrow compartment. This strategy has been shown to increase event-free survival and overall survival in comparison to chemotherapy alone (Child et al. 2003). However, the efficacy of this approach relies on the collection of an adequate number of hematopoietic stem and progenitor cells (HSPCs) prior to the damaging chemotherapy process. Unfortunately, even with granulocyte colony-stimulating factor (G-CSF) treatment, an agent often used for HSPC mobilization to the peripheral blood, where they can then be collected for transplantation, around 40–50% of MM patients are still unable to obtain optimal cell numbers for successful ASCT (Demirer et al. 1996, DiPersio et al. 2009).

Therefore, methods of improving the effectiveness of HSPC harvesting are required. Recently, clinical trials have been conducted to look at the efficacy of motixafortide, a selective inhibitor of CXCR4, for MM patients. The interaction between CXCR4 and its ligand CXCL12 has been shown to be crucially involved in the retention of HSPCs in the bone marrow. Here, we discuss how Crees et al. (2023) used the power of flow cytometry to assess the value of motixafortide treatment combined with G-CSF in MM patients before ASCT in a randomized phase 3 trial.

Motixafortide Treatment Enhances the Collection of HSPCs

This phase 3 trial randomized 120 patients to receive either motixafortide and G-CSF or placebo and G-CSF treatments prior to assessing their ability to reach the primary endpoint, a collection goal of at least six million HSPCs per kg of body weight within two blood collection days.

This study used the ZE5 Cell Analyzer to identify CD34+ HSPCs. The ZE5 Cell Analyzer can detect rare events like HSPCs quickly and without losing data due to electronic aborts, making it ideal for this type of research. Crees and colleagues found that while baseline levels of peripheral blood CD34+ HSPCs were similar before treatment, these numbers varied dramatically between the groups after motixafortide or placebo administration, with a median of 116 CD34+ HSPCs/μl in the peripheral blood of those receiving motixafortide plus G-CSF compared to 19 HSPCs/μl in those receiving placebo plus G-CSF. This manifested in an increase in patients meeting the primary endpoint of the study, with 92.5% of motixafortide and G-CSF–treated patients reaching the goal versus 26.2% of placebo and G-CSF–treated patients. Strikingly, 88.8% of motixafortide and G-CSF–treated patients met the secondary endpoint by reaching the cell count goal within one day of blood collection; only 9.5% of the controls met the same endpoint.

Motixafortide Treatment Is Safe and Reduces Healthcare Burden

Not only was the efficacy of motixafortide treatment measured but also its safety and healthcare resource utilization. The researchers looked at the severity of any treatment-emergent adverse effects (TEAEs), which were classified as adverse effects corresponding with the first dose of G-CSF through to 30 days after the final blood collection or the first chemotherapy dose, whichever took place first. While they found that around 93.8% of patients receiving motixafortide treatment reported a TEAE, the most common issues were transient, low-grade reactions, typically involving the local injection site, such as pain, erythema, and pruritis, or systemic effects, such as urticaria, flushing, and pruritis. No grade 4 TEAEs, which are characterized as life threatening or disabling events, or deaths occurred during this phase, indicating that motixafortide and G-CSF treatment is safe and generally well tolerated.

Additionally, the motixafortide treatment arm required an average of 5.26 injections of G-CSF and 1.23 blood collections per patient for optimal HSPC mobilization compared to 8.12 injections and 3.24 blood collections in the placebo group. Therefore, treatment with the combination of motixafortide and G-CSF requires fewer healthcare resources than G-CSF treatment alone.

Motixafortide Treatment Induces Pan-Mobilization of HSPC Subsets

Recently, increasing awareness has been brought to the immunophenotypic heterogeneity of HSPCs. The ZE5 Cell Analyzer, which can be equipped with up to five lasers and 30 detection parameters, is ideally suited to complex immunophenotyping, enabling Crees and colleagues to perform a multicolor immunophenotyping analysis on CD34+ HSPCs isolated from the initial blood collection of patients treated with placebo plus G-CSF, motixafortide plus G-CSF, or plerixafor (a low-affinity, short-acting CXCR4 inhibitor) plus G-CSF, to assess the impact these treatment regimens had on the constitution of the HSPC population. They found nine distinct subpopulations, which ranged from primitive stem cells displaying long-term self-renewing properties and multilineage potential to lineage-committed progenitor cells.

Motixafortide plus G-CSF treated individuals showed an increase in the percentage of common lymphoid progenitor (CLP), basophil precursor (BP), and natural killer cell precursor (NKP) subsets in comparison to the placebo plus G-CSF treatment group. Out of the nine subsets identified, eight showed significant increases in absolute numbers in the motixafortide group compared to the placebo controls, with over ten fold more primitive HSPCs. When the motixafortide and plerixafor treated cohorts were compared, motixafortide plus G-CSF induced significantly higher absolute numbers of multipotent progenitor (MPP), common myeloid progenitor (CMP), BP, and CLPs than plerixafor plus G-CSF administration. Overall, these data indicate that motixafortide therapy in combination with G-CSF stimulates pan-mobilization of HSPC subsets, including increases in primitive HSPCs and those capable of extensive reconstitution of multiple hematopoietic lineages.

Study Summary

For ASCT to be effective in the fight against MM, adequate numbers of HSPCs must be collected from the patient’s peripheral blood. As a mobilization agent, G-CSF alone is ineffective around 40–50% of the time. In this study, Crees and colleagues used Bio-Rad’s powerful ZE5 Cell Analyzer to show that combined treatment with the selective CXCR4 inhibitor motixafortide and G-CSF mobilizes HSPCs more than G-CSF treatment alone. Moreover, this novel treatment regimen was shown to be safe and resulted in less burden on healthcare resources.

Studying HSPCs with the ZE5 Cell Analyzer

This study by Crees et al. relied on the investigators’ ability to measure HSPCs in patients’ blood using flow cytometry. They used Bio-Rad’s ZE5 Cell Analyzer, which has a growing reputation in the field of HSPC analysis, to quantify the numbers and proportions of nine individual CD34+ HSPC subsets in 48 patients following HSPC mobilization. The ZE5 Cell Analyzer was also used by Lopez de Lapuente Portilla et al. (2022) to measure CD34+ cell levels as part of a genome-wide association study published in Blood.

The ZE5 Cell Analyzer is particularly suited to rare event analysis due to its high event rate, which it achieves without data or resolution loss. It also benefits from superior fluidics, integrated sample shaking and cooling, and automated sample acquisition, meaning high volumes of data can be collected quickly with minimal operator handling and without down time due to blockages.

Visit our website to see more publications featuring the ZE5 Cell Analyzer in cancer research. 


Albagoush SA et al. (2023). Multiple Myeloma. In StatPearls [Internet] (Treasure Island, Florida: StatPearls Publishing)., accessed October 8 2023.

Child JA et al. (2003). High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med 248, 1,875–1,883.

Crees ZD et al. (2023). Motixafortide and G-CSF to mobilize hematopoietic stem cells for autologous transplantation in multiple myeloma: a randomized phase 3 trial. Nat Med 29, 869–879.

Demirer T et al. (1996). Factors influencing collection of peripheral blood stem cells in patients with multiple myeloma. Bone Marrow Transplant 17, 937–941.

DiPersio JF et al. (2009). Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma. Blood 113, 5,720–5,726.

Lopez de Lapuente Portilla et al. (2022). Genome-wide association study on 13 167 individuals identifies regulators of blood CD34+cell levels. Blood 139, 1,659–1,669.

Rajkumar SV. (2022). Multiple Myeloma: 2022 update on diagnosis, risk-stratification and management. Am J Hematol 97, 1,086–1,107.

Previous post

Explore the Epigenomic Landscape of Single Cells with the scATAC You Seq

Next post

The Pioneer Antibody Discovery Platform: Clinical-Quality Lead Candidates in Industry-Leading Timelines