Chick embryo xenograft model of multiple myeloma recapitulates immune response, systemic dissemination, and sensitivity to anti-myeloma therapeutics Free

Introduction Multiple myeloma (MM) is an incurable plasma cell cancer comprising ~2% of cancer diagnoses and 10% of hematologic malignancies in the US. Current preclinical models are often constrained by high costs and extended experimental timelines. To address these limitations, we developed and validated a chick embryo chorioallantoic membrane (CAM) xenograft model for MM using clinically-relevant therapies. We investigated stromal cell support to tumor growth, examined the potential for systemic dissemination, patient-derived xenografting and immune responses elicited by MM cells. Collectively, these studies support the use of the CAM model for evaluating MM tumor-host interactions in a cost- and time-efficient manner.

Methods

Fertilized hen eggs were incubated at 37^oC on Embryonic Development Day (EDD) 0. On EDD3 some albumin was withdrawn, and a dorsal window was exposed. On EDD9, human MM cell lines ± HS5-stromal cells or CD138+ plasma cells from MM-patient bone marrow biopsies were implanted onto the CAM. After 3 days of tumor growth, clinically-used drugs Bortezomib (BTZ) and Lenalidomide (LEN), or vehicle, were topically applied daily. On EDD16, tumor xenografts were excised and stained for CD138, and organs harvested. Slides were digitally scanned at 400x and tumor burden was quantified using the HALO-AI platform (Indica Labs). gDNA was isolated from chick livers and qPCR for human-specific Alu sequences performed to detect systemically disseminated tumor cells. cDNA from chick spleen and intestine was assessed by qPCR for immune-related genes.

Results Implantation of high-risk MM cell lines JJN3 and KMS18 yielded macroscopic tumors in >90% of embryos (~5x5x3mm, ~8x8x3mm; 10⁶ cells/implant; n=82 embryos). Standard-risk lines (RPMI8226, U266, MM1S) also formed tumors in >95% of embryos, though markedly smaller (~3x2x2mm). JJN3 or KMS18 tumors co-cultured with HS5 stromal cells exhibited enhanced vascularization (n=57/60). Co-culture with HS5 cells led to increased tumor volumes (ellipsoid formula), revealing an ~118% increase for JJN3+HS5 (85.8 mm³) and a ~48% increase in KMS18+HS5 tumors (148.6 mm³), compared to monocultured JJN3/KMS18 tumors. MM patient-derived CD138+ cell xenografts (n=8) were successfully established in this model using as few as 2.24×10⁵ cells/implant.

Digital quantification of CD138+ plasma cells in JJN3 and KMS18 tumors treated with 0.6μg BTZ (across EDD12 and EDD14) showed a trend toward reduced tumor burden (JJN3: 86.14%; KMS18: 85.6% reduction; Mann-Whitney U testing p=0.1; n=3 experiments). BTZ±LEN also reduced tumor size in both JJN3+HS5 (n=3) and KMS18+HS5 (n=3) compared to DMSO controls.

In support of these data, human-specific Alu (hAlu) sequence qPCR was performed on EDD16-harvested livers to assess dissemination of MM tumor cells (JJN3 and KMS18) implanted onto the CAM. BTZ-treated tumors (JJN3/KMS18) had significantly lower hAlu signals compared to DMSO controls, consistent with reduced tumor dissemination to distant sites (p<0.05 by unpaired t-test; n=3). JJN3/HS5 co-cultured tumors exhibited increased dissemination to chick liver (median fold change in hAlu sequence detection: 0.33, range: 0.025-0.901, n=8) compared to JJN3 tumors alone (median: 0.046, range: 0.017-0.113, n=7).

Finally, qPCR of tumor-bearing spleens revealed BTZ-induced upregulation of TNF-α, IL-1b, IL-6, and Granzyme A transcripts, indicating enhanced systemic inflammatory and cytotoxic responses compared to DMSO controls. In contrast, intestinal tissues from BTZ-treated xenografts showed downregulation of TNF-α, Perforin, Granzyme A, and IL1b transcripts, alongside IL6, CXCLi2, and CD83 upregulation of 50-70%, suggesting a more immune-reactive microenvironment. These results highlight tissue-specific immune modulation in response to proteasome inhibition.

Conclusion This study validates the CAM model as a cost-effective, fast, and 3R-compliant system for studying MM progression and treatment response. It efficiently establishes human CD138+ xenografts offering a highly reproducible alternative to cell line-based animal models. The model replicates MM growth and therapy sensitivity and confirms stromal cells' pro-tumorigenic role. Current work explores its immune repertoire and potential for immune profiling, especially in the context of T-cell therapies and CAR-T/NK approaches. The feasibility of the approaches suggests a promising future for this model in preclinical MM precision medicine testing.